*Corresponding Author E-mail: lloyj@essex.ac.uk

© 2001 Association of Applied Biologists

Ann. appl. Biol. (2001), 138:111-115Printed in Great Britain 111

Identification of mutants in phosphorus metabolism

By JULIE C LLOYD*, OKSANA V ZAKHLENIUK and CHRISTINE A RAINES

Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK

(Accepted 23 January 2001; Received 13 September 2000)

Summary

Phosphorus availability is often limiting for plant growth. However, little is known of the pathwaysand mechanisms that regulate phosphorus (P) uptake and distribution in plants. We have developed ascreen based on the induction of secreted root acid phosphatase activity by low-P stress to identifymutants of Arabidopsis thaliana with defects in P metabolism. Acid phosphatase activity was detectedvisually in the roots of A. thaliana seedlings grown in vitro on low-P medium, using the chromogenicsubstrate, 5-bromo-4-chloro-3-indolyl-phosphate (BCIP). In low-P stress conditions the roots of wild-type plants stained blue, as the induced root acid phosphatase cleaved BCIP to release the colouredproduct. Potential mutants were identified as having white, or pale blue, roots under these conditions.Out of approximately 79 000 T-DNA mutagenised seedlings screened, two mutants with reduced acidphosphatase staining were further characterised. Both exhibited reduced growth and differences intheir P contents when compared to wild-type A. thaliana. The mutant with the most severe phenotype,pho3, accumulated high levels of anthocyanins and starch in a distinctive visual pattern within theleaves. The phenotypes of these mutants are distinct from two previously identified phosphorus mutants(pho1 and pho2) and from an acid phosphatase deficient mutant (pup1) of A. thaliana. This suggestedthat the screening method was robust and might lead to the identification of further mutants with thepotential for increasing our understanding of P nutrition.

Key words: Acid phosphatase, Arabidopsis thaliana, mutant, phosphorus nutrition

Introduction

Phosphorus (P) is an essential macronutrient forplant growth and is also a key regulator of manybiochemical processes (Marschner, 1995). Rootstake up inorganic phosphate from the soil, butunfortunately the availability of this ion is oftenlimiting, even in soils with a high P content(Marschner, 1995). Studies on the kinetics ofphosphate uptake, the transport of phosphatebetween roots and shoots, and the subcellulardistribution of the phosphate ion revealed thatphosphate metabolism is highly regulated within theplant (Bieleski, 1973). This primary regulation of Pdistribution that occurs continuously betweenorgans, and at a subcellular level between organellarcompartments, is an essential, constitutive process(Mimura, Sakano & Shimmen, 1996). It includes theregulation of P uptake rates which increase rapidlyin plants whose P supply has been withdrawn(Clarkson & Luttge, 1991), and inducible P�scavenging� systems, such as secretion of protonsand organic acids from roots and the production ofphosphatases and RNases (Lipton, Blanchar &Blevins, 1987; Duff, Sarath & Plaxton, 1994; Bariolaet al., 1994). In addition a range of secondaryresponses characteristic of (but not all specific to) Pstress are observed if plants become P deficient,

including anthocyanin accumulation, reducedgrowth rates and starch accumulation (Marschner,1995).

As yet little is known of the pathways andmechanisms that regulate P uptake and distributionin plants and the aim of more recent work has shiftedto unravelling the primary cellular and molecularprocesses that allow plants to control the utilisationof phosphate, and to respond to fluctuations in itsavailability (recently reviewed in Lynch & Deikman,1998). Some progress has been made towards thisby the cloning of phosphate transporters (Muchhal,Pardo & Raghothama, 1996) and studies on theeffects of phosphate starvation on gene expression(Liu et al., 1998) and metabolism (Plaxton &Carswell, 1999). It is possible to draw many parallelsbetween plant responses to phosphate deficiency andthose of microorganisms, for example the secretionof acid phosphatases and the expression of highaffinity phosphate transporters. In yeast the cellularsignalling system for perceiving phosphatedeficiency (the PHO system) has been studied foraround 20 years, using mutants and more recentlyapplying molecular genetics and cell biology to theproblem, and is now well characterised (Lenburg &O�Shea, 1996). Similarly, in bacteria the pho regulonwas elucidated using mutants (Torriani, 1990). Inplants, as in other organisms, mutants can provide a

112 JULIE C LLOYD, OKSANA V ZAKHLENIUK AND CHRISTINE A RAINES

powerful approach to dissecting the mechanismsinvolved in the P nutrition of plants.

Two A. thaliana mutants in P nutrition have beendescribed previously in the literature, both of whichwere identified by measuring leaf P content directly.pho1 has normal levels of P in roots but the shootsare severely P deficient (Poirier, Thoma,Sommerville & Schiefelbein, 1991; Delhaize &Randall, 1995), whereas pho2 accumulatesabnormally high leaf P levels (Delhaize & Randall,1995; Dong, Rengel & Delhaize, 1998). As analternative to the direct measurements of P contentused to isolate the pho1 and pho2 mutants, inductionof root acid phosphatase (APase) activity in responseto low-P stress was proposed as an approach toisolate plant mutants in P nutrition (Goldstein,Baertlein & Danon, 1989). Recently an A. thalianamutant (pup1) lacking one inducible APase isoformwas isolated using this screening method (Trull &Deikman, 1998). In this paper we describe the useof a modified version of this mutant screeningapproach to isolate two novel P mutants of A.thaliana.

Materials and Methods

Mutant screening and plant growthMutant screening was carried out using T-DNA-

tagged Arabidopsis thaliana (L.) Heynh (ecotypeWassilewskija) (Feldmann, Marks, Christianson &Quatrano, 1989). Seeds were surface-sterilised andcold-treated (at least 2 days at 4°C) prior to sowingon a low-Pi agar medium, containing half-strengthMS salts (lacking Pi), MS vitamins, 1 mg litre-1

glycine, 0.5 g litre-1 MES, 30 g litre-1 sucrose, 9 glitre-1 agar. The Pi content of this low-P media was11.31 ± 0.82 mM. Seeds were plated in rows and

plates were held in a vertical position in a growthcabinet (16 h photoperiod, 24°C). The roots of 7-8day old plants were overlaid with 6 g litre-1 agar thatcontained 0.1 g litre-1 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) and 1-3 days later the plants werescored for intensity of acid phosphatase staining inthe roots. Putative mutants with white or pale blueroots were transferred to fresh medium,supplemented with 0.63 mM PO

43-, and grown for a

further 7-10 days before transfer into soil. Plantswere allowed to self-pollinate and their progeny(100-200 seeds per individual plant) re-screened toconfirm the mutant phenotype. Phenotypic analysiswas carried out on homozygous plants that had beenselfed through three generations.

For studies on mature tissue, plants weregerminated and grown in the agar medium describedabove (supplemented with Pi) for 7 days, beforetransfer to compost (Fisons Levington, F2) andfurther growth with a 16 h day length at 24°C.

Phosphate analysisTotal P and Pi in plant samples was determined

using the methods described by Poirier et al. (1991).

Starch stainingLeaves were immersed in ethanol for 6 h to

remove pigments and stained with iodine (10 g litre-1)for 30 min to visualise the starch granules.

Results

The screening approach developed to identifyArabidopsis thaliana mutants with aberrant Pmetabolism was based on induction of secreted rootacid phosphatase activity by low-P stress. Thecolourless acid phosphatase substrate, BCIP, was

Fig. 1. Mutagenised seedlings were screened for root acid phosphatase activity when grown in agar medium lackingsupplementary Pi. A, Three rows of mutant seedlings were screened on each agar plate. After 7 days growth an agaroverlay, containing the substrate BCIP, was applied and plants were assessed for staining levels 1-2 days later. B,Magnified view (´4) of individual seedling showing root staining.

113Phosphorus mutants of Arabidopsis thaliana

P an

d P i ,

µm

ol/g

FW

0

20

40

60

80P Pi

S RS RFig. 3. Total phosphate (P) and inorganic phosphate(Pi) content of wild-type (WT; open bars) A. thalianaand the pho3 mutant (filled bars). P and Pi weremeasured in shoots (S) and roots (R) of 11-12 day oldseedlings grown in agar supplemented with 0.63 mMphosphate. Values represent the mean of 7-8measurements ± SD.

did not show similar signs of pigment accumulation,although the leaves had a pale appearance thatsuggested a possible reduction in photosyntheticpigment.

Measurements of the total P and Pi content of 11to 12 day old pho3 seedlings grown in vitro, inmedium supplemented with Pi, revealed that theshoots of pho3 plants contained reduced amountsof P and Pi compared to wild-type (Fig. 3). In rootsthe level of total P was also reduced in pho3 whencompared to wild-type, although no obviousdifference in Pi levels was evident in this tissue.

The phenotype of pho3 was also compared withthe previously described A. thaliana P mutants, pho1and pho2 (Poirier et al., 1991; Delhaize & Randall,1995), and also the pup1 mutant (Trull & Deikman,1998). Under these growth conditions the phenotypeof pup1 was indistinguishable from wild type A.thaliana (Fig. 4). In contrast, pho1, pho2 and pho3all had reduced growth rates, with the growth of pho1and pho3 more severely affected (Fig. 4). The leavesof pho1 were darker green than wild type A. thalianaand the other mutants, including pho3. Althoughsome purple pigmentation was observed in pho1 thiswas predominantly in the petiole and the flowerstems and was absent from the leaf margins.

Agar-grown seedlings of the pho3 mutant hadpreviously been shown to accumulate starch, evenwhen grown in P-sufficient conditions. To extendthis analysis the leaves of soil grown wild type A.thaliana, and of the P mutants were examined bystarch staining. Under these conditions wild type A.thaliana accumulated a small amount of starch,visible as a blue/black colour in the leaf (Fig. 5).The pho1 and pho2 mutants also showed only faintstaining, indicating low levels of starch. However,the leaves of pho3 had an intense colouration, further

applied in an agar overlay to the roots of plantsgerminated and grown for 7 days in agar mediumcontaining no added Pi. When wild-type plants weregrown with an adequate supply of P, roots remainedwhite in the presence of the BCIP overlay. However,in low-P stress conditions the roots of wild typeplants stained blue, as the induced root acidphosphatase cleaved BCIP to release the colouredproduct (Fig. 1A). Potential mutants were identifiedas having white, or pale blue, roots under theseconditions (Fig.1B). Out of approximately 79 000T-DNA mutagenised seedlings screened, almost 200were selected on the basis of reduced staining withBCIP. Two of these mutants, designated pho3(Zakhleniuk, Raines & Lloyd, 2001) and pho4, wereselected for further characterisation having showna consistent phenotype in subsequent generations.

Both pho3 and pho4 showed clear phenotypicdifferences to the wild type A. thaliana. Growth ofthe mutants in soil was reduced when compared towild type, with small stature and reduced leaf size(Fig. 2). There was also a delay in flowering ofbetween 2 (pho4) and 4 wk (pho3) when comparedwith wild-type. The leaf colour of pho3 wasstrikingly different to wild type; as leaves matureda band of purple anthocyanin pigmentation appearedaround the leaf margin and gradually spread towardsthe mid-vein as the leaves continued to age. pho4

WT pho4 pho3

Fig. 2. Growth of wild-type (WT) A. thaliana comparedwith two mutants, pho4 and pho3. Plants are shown atthe same developmental stage, aged 7, 8 and 10 wkrespectively.

P an

d P

i, µ

M/g

FW

S R S R

114 JULIE C LLOYD, OKSANA V ZAKHLENIUK AND CHRISTINE A RAINES

differentiating it from pho1 and pho2 and confirmingour earlier findings that high levels of starchaccumulate in this mutant (Zakhleniuk et al., 2001).Staining for starch in successive mature rosetteleaves from wild type A. thaliana and the pho3mutant revealed a distinctive pattern of starchaccumulation within the leaf, predominantly in theleaf margins and with a starch-free area towards theleaf base. The area staining intensely for starchcorresponded closely with the parts of the leafcontaining high levels of anthocyanin pigments.

Discussion

A screening protocol was developed to isolatemutants with altered P metabolism. The rationalebehind this approach was that a defect in theregulation of P metabolism could involve aberrantregulation of root low-P inducible acid phosphataseactivity. Putative mutants, which were unable toinduce the expression of their secreted root acidphosphatase in low-P medium, were identified usingthe substrate 4-bromo, 3-chloro indolyl phosphate(BCIP). An approach to isolate mutants in Pmetabolism based on the use of BCIP was firstdescribed by Goldstein et al. (1989), and involvedthe germination of seedlings on agar containing thisacid phosphatase substrate. Only mutants unable toinduce acid phosphatase activity under the low-Pconditions survived, since the product of the reactionis toxic. This screen successfully led to theidentification of the pup1 mutant, that lacks an acidphosphatase isozyme but has an otherwise wild-typephenotype (Trull & Deikman, 1998). In amodification of this method the BCIP substrate wasapplied in an agar overlay to 7 day-old seedlings.This avoided the toxicity evident when seedlingswere germinated in media containing BCIP and

potential mutants were instead selected on the basisof staining intensity of the root 24-48 h followingapplication.

Further analysis of two of the mutants identified,pho3 and pho4, revealed that both had pleiotropicphenotypes, as might be expected of mutants in Pnutrition. In addition to changes in P and Pi contentcompared to wild type A. thaliana both mutants werefound to have retarded growth rates and delayedflowering. The growth of pho3 was more severelycompromised than would have been expected fromthe level of Pi. One possible explanation for thisobservation might lie in the subcellular distributionof Pi within the pho3 mutant. A severe phenotypemight result from an inability to redistribute vacuolarreserves of Pi, that could lead to low cytosolic Piconcentrations while the total cellular Pi contentremained close to wild type. The high levels of starchobserved in the leaves of the pho3 mutant couldresult from a reduction in export of photosynthatefrom the chloroplast caused by low cytosolic Piconcentration. In order to resolve this question therelative sizes of cytosolic and vacuolar Pi pools willbe measured using 31P-NMR spectroscopy (Ratcliffe,1994).

Comparison of the growth and appearance of pho3and pho4 with previously identified P mutants, pho1and pho2 (Poirier et al., 1991; Delhaize & Randall,1995), clearly demonstrated that this screen hadsuccessfully isolated distinct mutants in P nutrition.

WT pho3 pho2 pho1

Fig. 4. Comparison of the growth of 7 wk-old soil-grown wild-type (WT) A. thaliana with the mutantspho1, pho2, pho3 and pup1.

WT pho1 pho2 pho3 pup1

WT

pho3

Fig. 5. Starch content of mature rosette leaves of wildtype (WT) A. thaliana and the mutants pho1, pho2 andpho3 (upper plate; actual size). Distinctive pattern ofstarch distribution in successive mature rosette leavesof pho3 (lower plate; magnification ´2). Plants weregrown in soil and harvested in the middle of the pho-toperiod. Starch was visualised by iodine staining.

WT pho3 pho2 pho1

115Phosphorus mutants of Arabidopsis thaliana

At present only two of the mutants identified havebeen partly characterised, and it is possible thatfurther novel mutants are among those awaitinganalysis. Mutants defective in P metabolism havethe potential to be valuable tools for investigatingthis important aspect of plant mineral nutrition. Along term goal of such work lies in its potentialpractical application in manipulating phosphateacquisition and metabolism. Ultimately this mightlead to more efficient fertiliser use, bringing botheconomic and environmental benefits.

Acknowledgements

This work was supported by the University ofEssex Research Promotion Fund and theBiotechnology and Biological Sciences ResearchCouncil (UK). We are grateful to the NottinghamArabidopsis Stock Centre for supplying T-DNAtagged seed for screening and thank Jill Deikmanand Manny Delhaize for gifts of mutant seed.Thanks also to George Ratcliffe (Department ofPlant Sciences, Oxford University) for helpfuldiscussions.

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