Differentiating phosphate-dependent and phosphate-independent systemic phosphate-starvation response networks in Arabidopsis thaliana through the application of phosphite.

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Cardiff School of Biosciences,
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Contents
FOCUS PAPERS: PHOTOSYNTHESIS IN VARIABLE ENVIRONMENTS
Preface
Johnson GN, Lawson, T, Murchie EH, Raines C.

Photosynthesis in variable environments

2371

Review papers
Schöttler MA, Tóth SZ, Boulouis A and Kahlau S. Photosynthetic complex stoichiometry dynamics in higher plants: biogenesis,
function, and turnover of ATP synthase and the cytochrome b6 f complex

2373

Dietz K-J.

2401

Eficient high light acclimation involves rapid processes at multiple mechanistic levels

Kaiser E, Morales A, Harbinson J, Kromdijk J, Heuvelink E and Marcelis LFM.
conditions
Allahverdiyeva Y, Suorsa M, Tikkanen M and Aro E-M.

Dynamic photosynthesis in different environmental
2415

Photoprotection of photosystems in luctuating light intensities

2427

Research paper
Retkute R, Smith-Unna SE, Smith RW, Burgess AJ, Jensen OE, Johnson GN, Preston SP and Murchie EH.
environments: does photosynthetic acclimation optimize carbon gain in luctuating light?

Exploiting heterogeneous
2437

RESEARCH PAPERS
Sun H, Li J, Song W, Tao J, Huang S, Chen S, Hou M, Xu G and Zhang Y. Nitric oxide generated by nitrate reductase increases nitrogen
uptake capacity by inducing lateral root formation and inorganic nitrogen uptake under partial nitrate nutrition in rice

2449

Girondé A, Poret M, Etienne P, Trouverie J, Bouchereau A, Le Cahérec F, Leport L, Orsel M, Niogret M-F, Deleu C and Avice J-C.
A proiling approach of the natural variability of foliar N remobilization at the rosette stage gives clues to understand the limiting
processes involved in the low N use eficiency of winter oilseed rape

2461

O’Brien M, Kaplan-Levy RN, Quon T, Sappl PG and Smyth DR. PETAL LOSS, a trihelix transcription factor that represses
growth in Arabidopsis thaliana, binds the energy-sensing SnRK1 kinase AKIN10

2475

Onoda Y, Schieving F and Anten NPR. A novel method of measuring leaf epidermis and mesophyll stiffness shows the ubiquitous
nature of the sandwich structure of leaf laminas in broad-leaved angiosperm species

2487

Jost R, Pharmawati M, Lapis-Gaza HR, Rossig C, Berkowitz O, Lambers H and Finnegan PM. Differentiating phosphate-dependent
and phosphate-independent systemic phosphate-starvation response networks in Arabidopsis thaliana through the application of
phosphite

2501

Li Q, Cao C, Zhang C, Zheng S, Wang Z, Wang L and Ren Z. The identiication of Cucumis sativus Glabrous 1 (CsGL1) required for
the formation of trichomes uncovers a novel function for the homeodomain-leucine zipper I gene

2515

Rasmann S, Chassin E, Bilat J, Glauser G. and Reymond P. Trade-off between constitutive and inducible resistance against
herbivores is only partially explained by gene expression and glucosinolate production

2527

Zhang Y, Wang Y, Taylor JL, Jiang Z, Zhang S, Mei F, Wu Y, Wu P and Ni J. Aequorin-based luminescence imaging reveals differential
calcium signalling responses to salt and reactive oxygen species in rice roots

2535

Riach AC, Perera MVL, Florance HV, Penield SD and Hill JK. Analysis of plant leaf metabolites reveals no common response to
insect herbivory by Pieris rapae in three related host-plant species

2547

Jin C, Fang C, Yuan H, Wang S, Wu Y, Liu X, Zhang Y and Luo J. Interaction between carbon metabolism and phosphate accumulation
is revealed by a mutation of a cellulose synthase-like protein, CSLF6

2557

Barbier F, Péron T, Lecerf M, Perez-Garcia M-D, Barrière Q, Rolčík J, Boutet-Mercey S, Citerne S, Lemoine R, Porcheron B, Roman H,
Leduc N, Le Gourrierec J, Bertheloot J and Sakr S. Sucrose is an early modulator of the key hormonal mechanisms controlling bud
outgrowth in Rosa hybrida

2569

Walter S, Kahla A, Arunachalam C, Perochon A, Khan MR, Scoield SR and Doohan FM.
both grain formation and mycotoxin tolerance

2583

Chai G, Kong Y, Zhu M, Yu L, Qi G, Tang X, Wang Z, Cao Y, Yu C and Zhou G.
roles in the regulation of secondary wall thickening and anther development
Xu C, Liu Y, Li Y, Xu X, Xu C, Li X, Xiao J and Zhang Q.
Boyer JS.

A wheat ABC transporter contributes to

Arabidopsis C3H14 and C3H15 have overlapping
2595

Differential expression of GS5 regulates grain size in rice

2611

Turgor and the transport of CO2 and water across the cuticle (epidermis) of leaves

2625

Dong L, Cheng Y, Wu J, Cheng Q, Li W, Fan S, Jiang L, Xu Z, Kong F, Zhang D, Xu P and Zhang S. Overexpression of GmERF5, a
new member of the soybean EAR motif-containing ERF transcription factor, enhances resistance to Phytophthora sojae in soybean

2635

Chateigner-Boutin A-L, Suliman M, Bouchet B, Alvarado C, Lollier V, Rogniaux H, Guillon F and Larré C.
reveals putative enzymes involved in cell wall metabolism in wheat grain outer layers

2649

Endomembrane proteomics

Continued overleaf

Porto DD, Bruneau M, Perini P, Anzanello R, Renou J-P, Santos HPD, Fialho FB and Revers LF.
requirement for bud break in apples: a putative role for FLC-like genes

Transcription proiling of the chilling
2659

Rojas-González JA, Soto-Súarez M, García-Díaz Á, Romero-Puertas MC, Sandalio LM, Mérida Á, Thormählen I, Geigenberger P,
Serrato AJ and Sahrawy M. Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and
important starch and metabolite changes in Arabidopsis thaliana

2673

Carrie C, Venne AS, Zahedi RP and Soll J.
peptidases in Arabidopsis thaliana

2691

Identiication of cleavage sites and substrate proteins for two mitochondrial intermediate

Zhang X, Wu Q, Cui S, Ren J, Qian W, Yang Y, He S, Chu J, Sun X, Yan C, Yu X and An C. Hijacking of the jasmonate pathway by the
mycotoxin fumonisin B1 (FB1) to initiate programmed cell death in Arabidopsis is modulated by RGLG3 and RGLG4

2709

Li J, Han Y, Liu L, Chen Y, Du Y, Zhang J, Sun H and Zhao Q.
length in upland rice

2723

qRT9, a quantitative trait locus controlling root thickness and root

Rosas-Santiago P, Lagunas-Gómez D, Barkla BJ, Vera-Estrella R, Lalonde S, Jones A, Frommer WB, Zimmermannova O, Sychrová H
and Pantoja O. Identiication of rice cornichon as a possible cargo receptor for the Golgi-localized sodium transporter OsHKT1;3

2733

Wang B, Li G and Zhang W-H.

2749

Brassinosteroids are involved in Fe homeostasis in rice (Oryza sativa L.)

Lu Y, Sasaki Y, Li X, Mori IC, Matsuura T, Hirayama T, Sato T and Yamaguchi J. ABI1 regulates carbon/nitrogen-nutrient signal
transduction independent of ABA biosynthesis and canonical ABA signalling pathways in Arabidopsis

2763

Deshpande GM, Ramakrishna K, Chongloi GL and Vijayraghavan U.
speciication and outgrowth

2773

Functions for rice RFL in vegetative axillary meristem

Jahan SN, Åsman AKM, Corcoran P, Fogelqvist J, Vetukuri RR and Dixelius C. Plant-mediated gene silencing restricts growth of the
potato late blight pathogen Phytophthora infestans

2785

Piazza A, Zimaro T, Garavaglia BS, Ficarra FA, Thomas L, Marondedze C, Feil R, Lunn JE, Gehring C, Ottado J and Gottig N.
The dual nature of trehalose in citrus canker disease: a virulence factor for Xanthomonas citri subsp. citri and a trigger for plant
defence responses

2795

Abu-Abied M, Rogovoy (Stelmakh) O, Mordehaev I, Grumberg M, Elbaum R, Wasteneys GO and Sadot E.
of microtubule behaviour in adventitious root induction

2813

Dissecting the contribution

Cover illustration: The cargo receptor Cornichon localizes at the endoplasmic reticulum. Co-localization of OsCNIH1 (magenta) with the
endoplasmic reticulum marker AtWAK2 (green) in leaf epidermis from Nicotiana benthamiana (chloroplasts in red). OsCNIH1 functions as a cargo
receptor for the sodium transporter OsHKT1;3 that appear to reside in the Golgi membrane (see Rosas-Santiago et al., pp. 2733–2748).

Journal of Experimental Botany, Vol. 66, No. 9 pp. 2501–2514, 2015
doi:10.1093/jxb/erv025 Advance Access publication 19 February 2015
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Differentiating phosphate-dependent and phosphateindependent systemic phosphate-starvation response
networks in Arabidopsis thaliana through the application of
phosphite
Ricarda Jost1,*, Made Pharmawati1,2, Hazel R. Lapis-Gaza1, Claudia Rossig1,
Oliver Berkowitz1,3,†, Hans Lambers1,4 and Patrick M. Finnegan1,4
1
2

4

*To whom correspondence should be addressed. E-mail: ricarda.jost@uwa.edu.au
Present address: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley
(Perth), Western Australia, Australia.

†

Received 18 November 2014; Revised 28 December 2014; Accepted 9 January 2015

Abstract
Phosphite is a less oxidized form of phosphorus than phosphate. Phosphite is considered to be taken up by the plant
through phosphate transporters. It can mimic phosphate to some extent, but it is not metabolized into organophosphates. Phosphite could therefore interfere with phosphorus signalling networks. Typical physiological and transcriptional
responses to low phosphate availability were investigated and the short-term kinetics of their reversion by phosphite, compared with phosphate, were determined in both roots and shoots of Arabidopsis thaliana. Phosphite treatment resulted in a
strong growth arrest. It mimicked phosphate in causing a reduction in leaf anthocyanins and in the expression of a subset
of the phosphate-starvation-responsive genes. However, the kinetics of the response were slower than for phosphate,
which may be due to discrimination against phosphite by phosphate transporters PHT1;8 and PHT1;9 causing delayed
shoot accumulation of phosphite. Transcripts encoding PHT1;7, lipid-remodelling enzymes such as SQD2, and phosphocholine-producing NMT3 were highly responsive to phosphite, suggesting their regulation by a direct phosphate-sensing
network. Genes encoding components associated with the ‘PHO regulon’ in plants, such as At4, IPS1, and PHO1;H1,
generally responded more slowly to phosphite than to phosphate, except for SPX1 in roots and MIR399d in shoots. Two
uncharacterized phosphate-responsive E3 ligase genes, PUB35 and C3HC4, were also highly phosphite responsive. These
results show that phosphite is a valuable tool to identify network components directly responsive to phosphate.
Key words: Arabidopsis thaliana, phosphate-starvation response, phosphate transport, phosphite, phosphonate, phosphorous
acid, phosphorus signalling networks, PSR genes, transcriptional regulation.

Introduction
Phosphite (H2PO3–, Phi) is a less oxidized form of phosphorus (P) than phosphate (H2PO4–, Pi). Phi is highly water soluble and less prone than Pi to adsorb to soil particles, which

makes it more accessible to plants (Ruthbaum and Baille,
1964). Phi competes with the essential macronutrient Pi for
uptake by plants, most probably through both high- and

Abbreviations: P, phosphorus; Phi, phosphite; Pi, inorganic phosphorus/phosphate/H2PO4–, PSR, phosphate-starvation-responsive.
© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Downloaded from http://jxb.oxfordjournals.org/ by guest on August 6, 2015

3

School of Plant Biology, The University of Western Australia, Crawley (Perth), Western Australia, Australia
Biology Department, Faculty of Mathematics and Natural Sciences, Bukit Jimbaran Campus, Udayana University, Bali, Indonesia
School of Veterinary and Life Sciences, Murdoch University, Murdoch, Western Australia, Australia
Institute of Agriculture, The University of Western Australia, Crawley (Perth), Western Australia, Australia

2502 | Jost et al.
with P-limited control plants (Carswell et al., 1996). While Phi
did not affect the total adenylate pool in P-limited Brassica
napus suspension cells in the same way as Pi, it did cause
changes in the in vivo phosphorylation status of a number of
proteins (Carswell et al., 1997). In A. thaliana, Ticconi et al.
(2001) observed that Phi prevented the induction of transcripts from the Pi-starvation-responsive (PSR) genes ACP5,
At4, and PT2 upon 14 d exposure of P-suficient seedlings to
a medium lacking Pi, but containing high concentrations of
Phi. The same plants showed reduced in vitro activities of PSR
ribonucleases RNS1 and RNS2 and of an acid phosphatase.
Within 1 d of transfer of P-suficient A.  thaliana seedlings
to a medium lacking Pi, Phi suppressed the typical root hair
formation and transcript accumulation of purple acid phosphatase PAP1 and Pi transporters PT1 and PT2 that occur
upon Pi withdrawal (Varadarajan et  al., 2002). Exposure
of A.  thaliana to Phi prevented not only PSR MGD2 and
MGD3 expression, but also changes in glycerolipid proiles
that accompany P-limited growth (Kobayashi et  al., 2006).
In P-limited tomato seedlings, Phi mimicked Pi in promoting proteolytic turnover of purple acid phosphatases (Bozzo
et al., 2004). In rice, long-term exposure (5–7 d) to Phi suppressed the Pi-starvation-induced expression of OsIPS1 and
OsIPS2 (Hou et al., 2005). In tobacco BY-2 cells, Phi caused
the reversion of autophagic protein turnover triggered by Pi
deprivation (Tasaki et al., 2014).
The irst evidence suggesting that Phi and Pi have discrete effects on P signalling networks came from work by
Stefanovic et  al. (2007), who showed that transcripts of
PHO1 and its close paralogue PHO1;H1 differentially
accumulated in plants treated with Pi or Phi. The PHR1dependent induction of PHO1;H1 under P-limiting conditions was attenuated by Phi, while the PHR1-independent
induction of PHO1 was not. This effect does not directly
depend on the MYB transcription factor PHR1, because,
unlike for PHO1;H1, the induction of another PHR1regulated paralogue, PHO1;H10, was not affected by Phi
(Ribot et al., 2008). Interestingly, both PHO1 and PHO1;H1
transcripts were less abundant in the P-limited pho2 mutant
and more strongly induced in the P-limited pdr2 mutant
compared with those in the wild type (Stefanovic et  al.,
2007). Disruption of the gene encoding endoplasmic reticulum (ER)-resident P5-type ATPase PDR2 affected local
Pi-sensing networks and heightened the sensitivity and
amplitude of metabolic responses to P limitation (Ticconi
et al., 2004). The conditional pdr2 short-root phenotype was
reversible by Phi. These observations strongly suggest that
Phi mimics Pi in local signalling networks, irrespective of
the plant’s P status.
Studies have so far addressed the question of whether Phi
can prevent the long-term accumulation of PSR gene transcripts. In this study, the question of whether the shorter
term kinetics of Phi suppression were similar to those of Pi
was addressed (Müller et al., 2004; Morcuende et al., 2007).
Organ-level accumulation of both Pi and Phi in P-limited
seedlings in A. thaliana accession Col-0 and three PHT1 transporter mutants was therefore determined. Root growth and
anthocyanin accumulation as well as gene expression proiles

Downloaded from http://jxb.oxfordjournals.org/ by guest on August 6, 2015

low-afinity transport systems (d’arcy-Lameta and Bompeix,
1991; Danova-Alt et  al., 2008). Phi uptake is strongly and
competitively inhibited in the presence of Pi (Pratt et  al.,
2009). Within the plant, Phi can be translocated, and it preferentially accumulates in sink tissues (Nartvaranant et  al.,
2004).
Phosphite was once abundant in the oceans, but it has been
oxidized over time (Pasek et al., 2013). Many microbes have
retained the ability to oxidize Phi to Pi, and even use it as
a reducing agent, namely for sulphate reduction (Poehlein
et al., 2013). Plants, however, are not able to metabolize Phi
(McDonald et al., 2001). Instead, P-limited plants are highly
sensitive to Phi and display toxicity symptoms such as leaf
chlorosis and stunted growth (McDonald et al., 2001; Ratjen
and Gerendas, 2009; Thao and Yamakawa, 2009). Other
detrimental effects caused by Phi are the arrest of primary
root growth, yellowing of the leaf lamina of young leaves,
and a patchy accumulation of anthocyanins in older leaves
(Varadarajan et al., 2002). Pratt et al. (2009) also showed that
respiration rates declined upon Phi treatment of P-limited
sycamore cells. It was recently found that the accumulation of
Phi impacts on metabolism in Arabidopsis thaliana, leading to
changes in the levels of several central metabolites (Berkowitz
et al., 2013).
Phi also triggers broad-spectrum resistance against pathogens with a (hemi)biotrophic lifestyle, such as oomycetes,
fungi, and nematodes (Smillie et  al., 1989; Hofgaard et  al.,
2010; Dias-Arieira et al., 2013; Percival and Banks, 2014). Phi
has been suggested to act as a priming agent of plant defence
responses in a number of plant–pathogen interactions
(Machinandiarena et al., 2012; Massoud et al., 2012; Dalio
et al., 2014). However, it is unclear how the primary recognition of Phi takes place, and which molecular pathways are
altered within the plant subsequently to induce this primed
state of heightened defence. Given that Phi is transported by
Pi transporters, these primary molecular interactions could
trigger changes in signal perception (Schothorst et al., 2013).
Phi accumulates in both the cytosol and organelles, while
the presence of Pi enhances Phi sequestration in the vacuole
(Danova-Alt et al., 2008). This is probably why plants with an
adequate P status can tolerate moderate Phi exposure without visible toxicity symptoms (Thao and Yamakawa, 2009).
Conversely, Phi inhibits the eflux of Pi from the vacuole,
which could exacerbate Pi-starvation symptoms (Pratt et al.,
2009) and lead to accelerated plant death (Singh et al., 2003).
Interestingly, the combined concentrations of Phi plus Pi
within roots and shoots of A. thaliana were remarkably constant, regardless of their ratio in the growth medium, demonstrating that plants sense both Pi and Phi and adjust their
uptake and allocation accordingly (Berkowitz et al., 2013).
Due to its physical similarity to Pi and non-metabolizable nature, Phi has been used as a tool to understand Pidependent signalling networks in plants. In several studies,
Phi in fact seemed to mimic Pi effectively. Brassica nigra
seedlings germinated on low-Pi media in the presence of high
(1–10 mM) Phi concentrations had reduced activation of Pistarvation-induced phosphoenolpyruvate phosphatase and
pyrophosphate-dependent phosphofructokinase compared

Short-term phosphite effects on phosphate signalling networks | 2503
in response to Phi treatment or Pi resupply were monitored in
P-limited Col-0 seedlings over a time-course from 1 d to 7 d.

Materials and methods

Root growth analysis and microscopy
After emergence of the radicle or transfer to a new plate, the position of the primary root tip was marked at 24 h intervals. Prior to
transfer or harvest, the seedlings were scanned at 600 dpi resolution
to determine root and root hair length, growth rate, and lateral root
number (LSM Image Browser v4.2; Carl Zeiss Microscopy GmbH,
Jena, Germany).
For microscopy (Axioplan Universal microscope; Carl Zeiss
Microscopy GmbH), roots were mounted onto slides in water under
glass cover slips. Images were electronically processed (AxioVision4;
Carl Zeiss Microscopy GmbH).
Metabolite quantiication
Fifteen volumes of 1% (v/v) acetic acid were added to frozen plant
powder (30–50 mg) and homogenized for three cycles of 45 s at
5000 rpm in the presence of two ceramic beads (ø 2 mm, Precellys
24 Tissue Disruptor; Bertin Technologies, Montigny-le-Bretonneux,
France). After incubation for 15 min on ice, the homogenization
process was repeated once. Cleared supernatants were used to

Relative quantiication of transcript abundance
mRNA was captured from tissue homogenates using oligo(dT)25coated magnetic beads (Dynabeads, Life Technologies Australia
Pty Ltd, Mulgrave, Australia) and converted to cDNA as previously
described (Jost et al., 2007). Aliquots of 0.5 ng of cDNA were ampliied in a 10 μl reaction volume containing 0.3 μM of each primer
and PCR master mix (Power SYBR® Green, Applied Biosystems,
Scoresby, Australia). Quantitative PCR and threshold cycle (Ct)
determination were performed using a luorescence baseline setting
of 0.3 (7500 FAST Real-Time PCR System, Applied Biosystems,
Scoresby, Australia). Data were normalized against PP2AA3 (formerly PDF2) and UBC9 reference genes (Czechowski et al., 2005).
PCR eficiencies for each primer pair were determined using the
LinReg algorithm (Ruijter et  al., 2009) (Supplementary Table S1
available at JXB online). Data were expressed either relative to normalized Ct values in control samples (ΔΔCt) or as 40–ΔCt values that
correlate with the relative transcript expression of the gene of interest (Bari et al., 2006). The detection limit of the assay was calculated
to be a 40–ΔCt value of 25.7 ± 0.1.
Statistical analysis
Statistically signiicant differences between treatments were determined using analysis of variance (ANOVA) and deined as P≤0.05
(SigmaStat v.  12.3, Systat Software Inc., San Jose, CA, USA).
Two-way ANOVA followed by Tukey’s post-hoc test was used
to separate means. Hierarchical clustering was performed using
squared Euclidean distance and complete linkage [J-Express 2012,
Norwegian Bioinformatics Platform and Norwegian Microarray
Consortium (http://www.molmine.com)] http://jexpress.bioinfo.no/
site/ (last accessed 27 January 2015) (Dysvik and Jonassen, 2001).

Results
Phosphite strongly reduced plant biomass production
A vertical growth system was used for A. thaliana Col-0 seedlings that allowed a direct comparison of the effects of Phi
versus Pi on the repression of Pi-starvation responses without the confounding effects of competition between Pi and
Phi. Using this system, P-limited seedlings were subjected
to continued Pi deprivation, Pi resupply, or Phi treatment.
Plant biomass did not differ signiicantly among the treatments within the irst 2 d of transfer (Fig.  1). After 3 d of
treatment, both the P-limited seedlings and those resupplied
with Pi had greater root and shoot biomass than seedlings at
days 1 and 2, while the biomass of the Phi-treated seedlings
was unchanged. Over the next 4 d, seedlings resupplied with
Pi recovered from P limitation with a proportional increase
in both root and shoot biomass that maintained the root-toshoot ratio at 0.30 ± 0.01. P-limited seedlings preferentially
allocated resources to roots over shoots, leading to a inal rootto-shoot ratio of 0.47 ± 0.03. Despite the greater partitioning
of biomass to roots, the root biomass after 7 d of further P

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Plant material and growth conditions
Seeds of A.  thaliana (L.) Heynh. Col-0 and homozygous T-DNA
insertion lines for pht1;1–2 (SALK 088568C) (Shin et  al., 2004),
pht1;8 (SALK 056529, Lapis-Gaza et al., 2014), and pht1;9-1 (SALK
050730) (Remy et al., 2012) were surface-sterilized for 2 min in 70%
(v/v) ethanol and 5 min in 5% (v/v) NaOCl, before being rinsed ive
times in sterile water. Seeds were resuspended in sterile 0.1% (w/v)
agar and stratiied in the dark for 24–48 h at 4 °C. Seedlings (12 per
plate) were grown vertically on 10 × 10 cm plates containing 50 ml of
nutrient solution [1 mM Ca(NO3)2, 2 mM KNO3, 0.5 mM MgSO4,
0.25 mM KH2PO4, 40 μM Fe-EDTA, 25 μM H3BO3, 2 μM MnCl2,
2  μM ZnSO4, 0.5  μM CuSO4, 0.075  μM (NH4)6Mo7O24, 0.15  μM
CoCl2, 50  μM KCl, pH 5.8] with 0.5% (w/v) 2-(N-morpholino)
ethanesulphonic acid and 1% (w/v) sucrose, and solidiied with
0.7% (w/v) agar (Plant TC Agar, cat.#A111, PhytoTechnology
Laboratories, Shawnee Mission, KS, USA). Plates were sealed
with 3M™ Micropore medical tape (Intouch Direct, Springwood,
Australia). Seedlings were grown in a 10/14 h day/night cycle with
200  μmol m–2 s–1 photosynthetically active radiation (PAR) at
21  °C (day), 19  °C (night), and 65% relative humidity. The plantavailable Pi present in the agar added another 5 μM to the medium.
This amount is within the range of Pi concentrations across gelling
agents (Jain et al., 2009). Preliminary experiments showed that concentrations of Pi ranging from 250 μM to 1 mM do not limit seedling growth in this system (data not shown). For the experiment,
seedlings grown on a medium with 250 μM Pi for 5 d were grown
for 4 d on plates without Pi supplementation (containing 250  μM
KCl instead) before being transferred to plates containing minimal
Pi (5 μM residual Pi in agar), or equimolar concentrations (250 μM)
of either Pi or Phi. The Phi solution was prepared from a fresh batch
of phosphorous acid (99%, Sigma Aldrich, Castle Hill, Australia) as
a ilter-sterilized 250 mM stock. The pH was adjusted to pH 5.8 with
KOH. There was