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Genetic and Genomic Evidence That Sucrose Is a GlobalRegulator of Plant Responses to Phosphate Starvationin Arabidopsis1[W][OA]

Mingguang Lei, Yidan Liu, Baocai Zhang, Yingtao Zhao, Xiujie Wang, Yihua Zhou,Kashchandra G. Raghothama, and Dong Liu*

Protein Science Laboratory of the Ministry of Education, School of Life Sciences, Tsinghua University, Beijing100084, China (M.L., Y.L., D.L.); State Key Laboratory of Plant Genomics and National Center for Plant GeneResearch, Institute of Genetics and Developmental Biology (B.Z., Y.Zho.), and State Key Laboratory of PlantGenomics, Institute of Genetics and Developmental Biology (Y.Zha., X.W.), Chinese Academy of Sciences,Beijing 100101, China; and Department of 2Horticulture and Landscape Architecture, Purdue University,West Lafayette, Indiana 47907 (K.G.R.)

Plants respond to phosphate (Pi) starvation by exhibiting a suite of developmental, biochemical, and physiological changes tocope with this nutritional stress. To understand the molecular mechanism underlying these responses, we isolated anArabidopsis (Arabidopsis thaliana) mutant, hypersensitive to phosphate starvation1 (hps1), which has enhanced sensitivity in almostall aspects of plant responses to Pi starvation. Molecular and genetic analyses indicated that the mutant phenotype is caused byoverexpression of the SUCROSE TRANSPORTER2 (SUC2) gene. As a consequence, hps1 has a high level of sucrose (Suc) inboth its shoot and root tissues. Overexpression of SUC2 or its closely related family members SUC1 and SUC5 in wild-typeplants recapitulates the phenotype of hps1. In contrast, the disruption of SUC2 functions greatly inhibits plant responses to Pistarvation. Microarray analysis further indicated that 73% of the genes that are induced by Pi starvation in wild-type plants canbe induced by elevated levels of Suc in hps1 mutants, even when they are grown under Pi-sufficient conditions. These genesinclude several important Pi signaling components and those that are directly involved in Pi transport, mobilization, anddistribution between shoot and root. Interestingly, Suc and low-Pi signals appear to interact with each other bothsynergistically and antagonistically in regulating gene expression. Our genetic and genomic studies provide compellingevidence that Suc is a global regulator of plant responses to Pi starvation. This finding will help to further elucidate thesignaling mechanism that controls plant responses to this particular nutritional stress.

Although plants require ample amounts of phos-phate (Pi) for their growth and development, theyoften encounter a Pi deficiency in their surroundingenvironments (Raghothama, 1999; Vance et al., 2003).Unlike animals, plants are sessile organisms, so theymust respond to this adverse condition through ad-justment in their growth and development and inmetabolic activities. Through the long evolution pro-cess, plants have developed sophisticated strategies tobetter adapt to Pi starvation (Yuan and Liu, 2008).

These strategies include changes in the root architec-ture system (i.e. reduction of primary root growth andthe formation of more lateral roots and root hairs),increased expression of Pi transporter genes, the in-duction and secretion of organic acid, RNase, andacid phosphatases (APases), and the accumulationof starch and anthocyanin. Although these adaptiveresponses have been widely studied and well docu-mented in a variety of plant species, the molecularmechanisms that regulate these responses are stilllargely unknown.

In the last decade, significant progress has beenmade in identifying signaling components that areinvolved in plant responses to Pi starvation (Yuan andLiu, 2008). PHOSPHATE STARVATION RESPONSE1(PHR1) is one of the most studied transcription factors;it encodes a MYB domain-containing protein thatbinds to a short DNA motif commonly found in thepromoters of several Pi starvation-induced (PSI) genes(Rubio et al., 2001). The mutation of PHR1 causesdefects in a subset of Pi starvation responses. Othertranscription factors that are involved in Pi responsesinclude WRKY75, WRKY6, ZAT6, MYB26, andBHLH32 in Arabidopsis (Arabidopsis thaliana) and

1 This work was supported by the Ministry of Science andTechnology of China (grant nos. 2007CB948200 and 2009CB119100),the Ministry of Agriculture of China (grant nos. 2009ZX08009–123Band 2008ZX08009–003), and theNatural Science Foundation of China(grant no. 30670170).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Dong Liu ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

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OsPTF1 and OsPHR2 in rice (Oryza sativa; Chen et al.,2009; Lin et al., 2009). In Arabidopsis, the siz1 mutantexhibits exaggerated Pi starvation responses (Miuraet al., 2005). SIZ1 is a SUMO E3 ligase that is localizedin the nucleus. In vitro sumoylation assay has shownthat PHR1 is the direct target of SIZ1. Besides severalPSI genes, PHR1 also controls the transcription ofmiRNA399. miRNA399 can directly bind to the 5#untranslated region of PHO2 mRNA and result in itsdegradation (Bari et al., 2006). The PHO2 gene encodesa ubiquitin E2 conjugation enzyme and is involvedin the regulation of plant Pi homeostasis (Bari et al.,2006; Chiou et al., 2006). Interestingly, the activity ofmiRNA399 can further be controlled through a mech-anism called “target mimicry” (Franco-Zorrilla et al.,2007). In Arabidopsis, AtIPS1 and At4 genes encodenon-protein-coding transcripts that function in the in-ternal translocation of Pi from shoot to root (Shin et al.,2006). Both of them contain a motif that is partiallycomplementary to miRNA399. The imperfect pairing ofAtIPS1/At4 with miRNA399 results in the sequestra-tion of miRNA399, thus altering the mRNA level of thePHO2 gene. Recently, some SPX domain-containingproteins have also been shown to act as importantregulators of Pi starvation responses in both Arabidop-sis and rice (Wang et al., 2009; Liu et al., 2010a).Sugar sensing and signaling are important regula-

tory components of plant growth and development aswell as metabolic activities (Rolland et al., 2006).Experimental evidence has suggested that sugar sig-naling may also be involved in plant responses to Pistarvation. Stem girdling of white lupin (Lupinus albus)to block the movement of photosynthates from shootto root severely affected the induction of PSI genes inPi-starved root (Liu et al., 2005). Similarly, it has beenobserved that the level of induction of PSI genes inArabidopsis seedlings is positively correlated with theconcentration of Suc present in the culture medium(Karthikeyan et al., 2007). Both groups have alsonoticed that light has a significant effect on the induc-tion of PSI genes (Liu et al., 2005; Karthikeyan et al.,2007). When plants are grown in the dark withoutaddition of Suc in the medium, the expression of PSIgenes is significantly reduced; however, if exogenousSuc is supplied in the dark, the expression of PSI genescan be sustained to a certain degree. The similar effectsof photosynthates on Pi starvation-induced clusterroot formation in white lupin and the expression ofmiRNA399 in common bean (Phaseolus vulgaris) havealso been observed (Zhou et al., 2008; Liu et al., 2010b).In contrast, the Arabidopsis pho3 mutant carries adefective copy of the SUCROSE TRANSPORTER2(SUC2) gene, leading to substantially reduced trans-port of Suc from shoot to root and the suppression ofinduction and secretion of APase on its root surface(Zakhleniuk et al., 2001; Lloyd and Zakhleniuk, 2004).In addition, earlier studies have revealed that theexpression of many genes involved in the synthesis,translocation, and degradation of Suc was alteredduring Pi starvation (Hammond et al., 2003; Vance

et al., 2003; Wu et al., 2003; Misson et al., 2005; Mulleret al., 2007). Furthermore, an increase in Suc biosyn-thesis in Pi-starved leaves has been observed in avariety of plants, including Arabidopsis, bean, barley(Hordeum vulgare), spinach (Spinacia oleracea), and soy-bean (Glycine max; Hammond and White, 2008). Anincrease in Suc concentration is thought to be an earlyresponse of plants to Pi starvation (Hammond andWhite, 2008). All these results suggested that Suc mayplay a signaling role during the plant response to Pistarvation. However, how broadly Suc can affect plantresponses to Pi starvation has not been examined, andthe question of how Suc acts at the molecular level totrigger multiple Pi responses remained to be an-swered.

In this work, we characterize an Arabidopsis mu-tant, hypersensitive to phosphate starvation1 (hps1), whichshowed enhanced sensitivity in almost all the testedparameters of plant responses to Pi starvation. Ourgenetic and molecular studies of this mutant, com-bined with genomic analysis, provide compelling ev-idence that Suc is a global regulator of plant responsesto Pi starvation.

RESULTS

Identification of the hps1 Mutant

The hps1mutant was identified from an ArabidopsisT-DNA activation library. This library was generatedby transforming a transgenic line bearing an AtPT2-LUC construct (herein referred as the wild-type plant)with a pSuperTag2 activation vector (Koiwa et al.,2006; Supplemental Fig. S1). In this construct, a fireflyluciferase (LUC) gene is fused to the promoter of theAtPT2 (AtPht1;4) gene, which encodes a high-affinityPi transporter (Karthikeyan et al., 2002). Pi deficiencyinduced the expression of the reporter gene fused tothe Pi starvation-responsive AtPT2 promoter. Whengrown under Pi starvation, the hps1 mutant displayedenhanced LUC expression compared with wild-typeplants (Fig. 1A). The enhanced expression of AtPT2-LUC in hps1 is rather restricted to the junction betweenroot and hypocotyl and the root tip. To confirm thatthe enhanced expression of AtPT2-LUC was due tothe mutation that occurred in a regulatory component,an AtPT2-GUS marker gene was introduced into hps1plants through a genetic cross. Consistent with theresults of AtPT2-LUC, expression of the AtPT2-GUSreporter gene was also greatly enhanced at thehypocotyl-root junction and root tip of hps1 (Fig. 1, Band C). Further examination indicated that enhancedAtPT2-GUS expression mainly occurred in lateralroots, while expression in the primary root was rela-tively suppressed compared with the wild type underthe no-phosphorus (P2) condition. In hps1 mutants,the expression of AtPT2-LUC and AtPT2-GUS wasevident even under Pi sufficiency (Fig. 1). This sug-gested a hyperinduction of AtPT2-LUC in the hsp1

Suc and Pi Starvation Responses

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mutant. To further ensure that the enhanced expres-sion of AtPT2-LUC and AtPT2-GUS in hps1 mutants isa true reflection of the changes in expression of theendogenous AtPT2 gene, a real-time PCR techniquewas used to measure the mRNA level of AtPT2 inwild-type and hps1 plants. When the AtPT2 geneexpression level of wild-type plants grown under thePi-sufficient condition was set as 1, we found that in-duction of AtPT2 by Pi starvation in wild-type andhsp1 mutant plants was 4.2- and 30-fold, respectively(Fig. 2). Even though hsp1mutant plants already had ahigh basal expression ofAtPT2 (2.5 comparedwith 1 ofthe wild type), it still showed a higher induction (12.5-fold) by Pi starvation than the wild type (4.2-fold).

When hps1 was back-crossed to a wild-type plant,the F1 progeny seedlings behaved like hps1. And theF2 progeny showed a 1:3 segregation of wild-type tomutant phenotypes (51 wild type versus 142 hps1). ThepSuperTag2 plasmid harbors a Basta-resistant geneand a superpromoter that is located next to the T-DNAright border (Supplemental Fig. S1). In the F2 popula-tion, all the wild-type seedlings were Basta sensitiveand mutants were resistant. These results indicatedthat the hps1 mutant phenotype is caused by a singledominant mutation, probably due to the T-DNA in-sertion.

The hps1 Mutation Enhances Pi Starvation-Induced

Gene Expression

AtPT1 is another major high-affinity Pi transporterin Arabidopsis whose expression is also highly inducedby Pi starvation (Muchhal et al., 1996; Karthikeyanet al., 2002). When hps1 mutation was introduced intothe transgenic line carrying an AtPT1-GUS constructthrough a genetic cross, the expression of AtPT1-GUSwas also highly induced under Pi starvation in thelateral roots around the hypocotyl-root junction andthe root tips (Supplemental Fig. S2). The enhancedinduction of the endogenous AtPT1 gene was con-firmed by real-time PCR analysis (Fig. 2).

In addition to AtPT1 and AtPT2, we examined theexpression of six other PSI genes that encode anotherPi transporter (PHT1;5), an APase (ACP5), an RNase(RNS1), two noncoding mRNAs of At4 and AtIPS1,and miRNA399D. Similar to AtPT1 and AtPT2, theinduction of these six PSI genes by Pi starvation wasalso dramatically enhanced in hps1 plants comparedwith wild-type plants (Fig. 2). In fact, even under Pi-sufficient conditions, the expression of these PSI genes(except AtPT1 and miRNA399D) in hps1 plants washigher than in wild-type plants. This indicated that thehps1 mutation alone is sufficient to induce some PSIgenes even without Pi starvation.

hps1 Overproduces APases, Anthocyanin, and Starchunder Pi Starvation

To respond to Pi starvation, plants increase theproduction and secretion of APases to mobilize inter-nal and external Pi. The secreted APases on the rootsurface can cleave 5-bromo-4-chloro-3-indolyl phos-phate (BCIP) to produce a blue precipitate (Zakhleniuket al., 2001). When BCIP was applied to the rootsurfaces of Pi-starved wild-type and hps1 seedlings,hps1 roots showed much darker blue staining than thewild type (Supplemental Fig. S3A). In gel-assay of totalextracted soluble proteins (for a detailed procedure,see “Materials and Methods”) showed an enhancedproduction of a major isoform of APase (approxi-mately 95 kD) in hps1 on both P+ and P2 media(Supplemental Fig. S3B). Consistent with the results ofBCIP staining and in-gel assay, the total APase activityis enhanced in the hps1 mutant compared with the

Figure 1. AtPT2-LUC and AtPT2-GUS expression in wild-type andhps1 seedlings. A, Seeds of wild-type (WT) and hps1 plants weredirectly sown on MS medium with (P+) or without (P2) supplementedinorganic Pi and grown for 8 d. Luciferin was sprayed on the surface of8-d-old seedlings, and photographs were taken either with a digitalcamera (top row) or a CCD camera (bottom row). B and C, Expressionof AtPT2-GUS in 8-d-old wild-type and hps1 seedlings. The GUSactivities at the hypocotyl-root junction (B) and the root tip (C) areshown.

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wild type, as shown by quantitative analysis using aspectrophotometer (Supplemental Fig. S3C).Other characteristic responses of plants to Pi star-

vation include anthocyanin and starch accumulation.When hps1 and wild-type plants were grown on P2medium for 2 weeks, the wild-type seedlings turnedonly light purple, whereas the whole shoot of the hps1mutant had already become dark purple (Fig. 3A).Quantitative analysis showed that, under Pi starva-tion, the anthocyanin content in the hps1 seedlings wasthree times as much as in wild-type plants (Fig. 3B). Inaddition, hps1 plants have reduced amounts of chlo-rophyll a and b and carotene under both Pi-sufficientand Pi-deficient conditions compared with wild-typeplants (Fig. 3C), suggesting a reduced photosynthesisactivity. Our following microarray analysis indicatedthat six genes involved in photosynthesis were down-regulated in hps1 mutants (Supplemental Table S1).The accumulation of starch in hps1 plants was alsomuch higher than in the wild type under both P+ andP2 conditions, as revealed by iodine staining (Fig. 3D).All of the above results indicated that the hps1 muta-tion affects multiple aspects of plant responses to Pistarvation in addition to the expression of PSI genes.

Ion Homeostasis Is Perturbed in the hps1 Mutant

To sustain normal growth and development, it isimportant for plants to have a mechanism to maintainion homeostasis, including Pi, as they respond toexternal stress (Raghothama, 1999). To examine theeffects of hps1 mutation on ion homeostasis in plantcells, we analyzed the accumulation of nine elementsin wild-type and hps1 plants under both Pi-sufficientand Pi-deficient conditions. When grown under Pi

starvation, the contents of cellular Pi and total phos-phorus in wild-type plants decreased dramatically inboth their shoot and root tissues (Fig. 4, A and B).Similarly, when hps1 plants were grown under Pistarvation, they accumulated less cellular Pi and totalphosphorus than on Pi-sufficient medium. However,hps1 plants contained more cellular Pi in roots and lessin shoots under both P+ and P2 conditions comparedwith wild-type plants (Fig. 4A), indicating that thehps1mutation caused a shift of allocation of Pi betweenshoots and roots. It has been reported that plantsgrown on Pi-deficient medium would accumulatemore iron (Hirsch et al., 2006). Our analysis confirmedthese observations and further showed that, on P2medium, hps1 plants accumulated even more iron(about 2-fold higher compared with wild-type plants)in both shoots and roots (Fig. 4C). Another interestingphenomenon is that wild-type plants accumulatedrelatively high amounts of zinc ions in their rootscompared with shoots on P+ medium (Fig. 4D). How-ever, differential accumulation of zinc in shoots wasnot obvious in hps1 plants.

Besides phosphorus, iron, and zinc, we also exam-ined the contents of sodium, potassium, calcium, mag-nesium, manganese, and sulfur in both wild-type andhps1 mutant plants under P+ and P2 conditions. Wefound that the concentrations of these ions were allmore or less perturbed in hps1 plants (SupplementalFig. S4).

The hps1 Mutation Affects Plant Root Development

In addition to plant responses to Pi starvation andion homeostasis, we also found that the hps1 mutationhad a marked effect on plant root development. When

Figure 2. Quantitative analysis of PSIgene expression in wild-type and hps1seedlings. Nine-day-old wild-type andhps1 seedlings grown on MS P+ or P2medium were used for real-time PCRanalysis. The PSI genes examined inthis analysis encode three high-affinityPi transporters (AtPT1, AtPT2, andPHT1;5), an APase (ACP5), an RNase(RNS1), two noncoding mRNAs(At4 and AtIPS1), and a microRNA(miRNA399D). Values are means 6 SE

of three biological replicates where thefold changes are normalized to tran-script levels in the wild type on P+medium. Asterisks indicate significantdifferences compared with the wildtype (P , 0.05, t test). White bars,Wild-type plants; black bars, hps1plants.





grown on P+ medium, the roots of wild-type plantsexhibited indeterminate growth during 15 d after seedgermination. At day 9, wild-type plants seldomformed lateral roots (Fig. 5A). During the same growthperiod, the length of primary roots of hps1 plants wasabout half that of wild-type plants. hps1 plants alsoformed numerous lateral roots near the junction be-tween hypocotyl and root (Supplemental Fig. S5). OnP2 medium at day 9, the growth of primary roots ofwild-type plants was strongly inhibited while severallateral roots were formed, as reported previously byanother group (Williamson et al., 2001). In hps1 plantsgrown under P2 conditions, overall growth, includingboth shoots and roots, was severely inhibited. Thelength of the lateral roots was much shorter comparedwith that of the wild type, although there was notmuch difference in the number of lateral roots (Fig. 5,A and B). The hps1mutation also had a strong effect onroot hair development. As anticipated, wild-typeplants grown on P+ medium developed few shortroot hairs close to the root tip and maturation zone(Fig. 5, B and C). When grown on P2 medium, boththe number and length of root hairs increased in thewild type. The hps1 plants formed many short roothairs near the root tip and maturation zone, evenunder Pi-sufficient conditions (Fig. 5, B and C). Whengrown under Pi starvation, the increase in root hairlength and density was less than that of the wild type.

Taken together, these data suggested that hps1 plantsdisplay a rather constitutive Pi starvation-induceddevelopmental response under Pi sufficiency and be-come more sensitive to Pi starvation-induced inhibi-tion of plant growth and development. When hps1mutant plants were grown in soil, however, there wasno obvious morphological difference compared withwild-type plants.

The hps1 Mutant Phenotype Is Caused byOverexpression of the SUC2 Gene

Since the hps1 mutant phenotype is tightly linked toT-DNA insertion, we cloned the T-DNA-flanking se-quence in hps1 plants. The T-DNA is inserted 170 bpupstream of the ATG start codon of the SUC2 gene(Fig. 6A). Because of the presence of a superpromotersequence next to the T-DNA right border (Koiwa et al.,2006), there was a likelihood of constitutive overex-pression of the SUC2 gene in hps1 plants. Indeed, themRNA level of SUC2 in hps1 was about 15-fold higherthan that of wild-type plants, as revealed by real-timePCR analysis (Fig. 6B). [14C]Suc uptake assay showedthat the Suc uptake rate in roots of hps1 plants wasabout 4-fold higher than in wild-type plants (Fig. 6C).(For a detailed procedure of measuring Suc uptakerate, see “Materials and Methods.”) Similarly, theaccumulation of Suc in both shoots and roots of hps1seedlings was much higher than that of wild-typeplants, especially in root tissues under P+ conditions(Fig. 6D). The high content of Suc in shoots of the hps1mutant was probably due to the transport of Suc from

Figure 3. Analyses of anthocyanin, chlorophyll, and starch contents inwild-type and hps1 seedlings. A, Morphological appearance of 14-d-old wild-type (WT) and hps1 seedlings grown on MS P+ and P2media.B, Anthocyanins were extracted from the shoots of 14-d-old wild-typeand hps1 seedlings grown on MS P+ and P2 media. The contents ofanthocyanin were determined spectrophotometrically and expressed asA530 mg21 fresh weight (FW). C, Chlorophylls and carotenes wereextracted from the shoots of 10-d-old wild-type and hps1 seedlingsgrown on MS P+ and P2 media. The contents of the pigments weredetermined spectrophotometrically. Asterisks indicate significant dif-ferences compared with wild-type plants (P , 0.05, t test). D, Starchaccumulation in 11-d-old wild-type and hps1 seedlings grown on P+and P2 media as revealed by iodine staining.

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root tissues with water through the xylem. We noticedthat the Suc levels in both shoot and root tissues ofwild-type plants were also elevated under Pi starva-tion (Fig. 6D). Since Suc is readily metabolized into Glcand Fru as it enters the metabolic pathway, we mea-sured the contents of these two monosaccharides inhps1 and wild-type plants. The amount of Glc and Fruin hps1 plants was less than half of that in the wildtype, except in Pi-starved roots, where the Glc levelwas about 25% lower (Supplemental Fig. S6). Tofurther confirm that the hps1 mutant phenotype wascaused by overexpression of the SUC2 gene, we intro-duced the SUC2 gene into the AtPT2-LUC line underthe control of a cauliflower mosaic virus 35S promoter.Ninety-two independent transgenic lines were gener-ated, and 90% of the lines showed the hps1 mutantphenotype (i.e. superinduction of the AtPT2-LUCgene, strong inhibition of primary root growth, for-mation of more lateral roots; Fig. 7A). In fact, most ofthese transgenic lines showed a stronger phenotypethan hps1, probably due to the higher SUC2 expressiondriven by the strong 35S promoter. In contrast, whengrown on Murashige and Skoog (MS) Suc-free me-dium in both P+ and P2 conditions for 14 d, there wasno obvious phenotypic difference between the wildtype and the hps1 mutant in terms of AtPT2-LUCexpression and seedling morphologies (SupplementalFig. S7). Taken together, these results demonstratedthat the hps1 mutant phenotype was indeed caused bythe overexpression of SUC2 that resulted in enhanceduptake of Suc from the culture medium.In the Arabidopsis genome, there are nine annotated

Suc transporter genes, which can be divided into threegroups based on their sequence similarity (Sauer,

2007). SUC2 is a plasma membrane-associated high-affinity Suc/proton symporter that is specifically ex-pressed in phloem companion cells and root vasculartissues (Stadler and Sauer, 1996). SUC1 and SUC5 arealso plasma membrane-associated Suc transportersand share high sequence homologies with SUC2.However, SUC1 is mainly expressed in floral tissueand developing seeds, whereas SUC5 expression isembryo specific. In the same subgroup as SUC2, SUC6and SUC7 are annotated as pseudogenes, and SUC8and SUC9 are expressed in floral tissues (Sauer et al.,2004). SUC3 and SUC4 belong to two different sub-groups. The SUC4 protein is localized in tonoplast,which is not involved in the phloem transport of Sucfrom source to sink tissues (Endler et al., 2006). SUC3has very low affinity to Suc and is mainly expressed insieve elements in sink tissues (Barker et al., 2000). Todetermine if the overexpression of SUC genes withdifferent functions has similar effects as SUC2, weintroduced SUC1, SUC5, SUC3, and SUC4 genes intoAtPT2-LUC plants under the control of the 35S pro-moter. As shown in Figure 7B, only the overexpressionof SUC1 and SUC5 had a similar effect as SUC2 onAtPT2-LUC gene expression and root development.This was probably due to the overexpression ofplasma membrane-associated high-affinity Suc trans-porters (SUC1, SUC2, and SUC5) leading to increaseduptake of Suc from culture medium, whereas SUC3and SUC4 were not able to fulfill this role. Thus, webelieve that it is the level of Suc, rather than the activityof a particular Suc transporter, that regulates the extentof plant responses to Pi starvation. In fact, the soil-grown hps1 and 35S-SUC1, 35S-SUC2, and 35S-SUC5plants did not show obvious phenotypic differences

Figure 4. Comparison of ion contentsbetween wild-type and hps1 plants.Plants were grown on MS P+ and P2media for 9 d. A, The cellular Pi wasmeasured using the Ames (1966)method. B to D, The element contentsof phosphorus (B), iron (C), and zinc(D) were determined by ICP-OES. As-terisks indicate significant differencescompared with the wild type (P ,0.05, t test). White bars, Wild-typeplants; black bars, hps1 plants. DW,Dry weight; FW, fresh weight.





compared with the wild type. This is probably due tothe absence of a high concentration of Suc in soils forthese plants to take up. This phenomenon furthersupported the notion that high Suc is critical for plantsto display hypersensitive responses to Pi starvation.

Reduction of Suc in Roots Severely Inhibits PlantResponses to Pi Starvation

To provide further genetic evidence that the accu-mulation of Suc is critical for plant responses to Pistarvation, we identified a Salk line (SALK_087046),designated as suc2-5, in which the T-DNA is insertedinto the first exon of the SUC2 gene (Fig. 6A; Supple-mental Fig. S8B). Reverse transcription (RT)-PCR anal-ysis of SUC2 gene expression indicated that suc2-5 is anull allele (data not shown). The suc2-5 plants showedsimilar growth characteristics in soil as reported pre-viously for other suc2 mutant alleles (i.e. severelyretarded growth with high accumulation of anthocy-anin in leaves; Gottwald et al., 2000; Supplemental Fig.

S8C). On sugar-free medium, suc2-5 seedlings havevery short roots (Supplemental Fig. S8A). To examinethe induction of APase activity and PSI gene expres-sion, the seeds of wild-type and suc2-5 plants weresown on MS P+ medium with Suc and grown for 5 dand then transferred toMS P2mediumwith no addedSuc for another 5 d. As shown in Figure 8A, the suc2-5mutant showed almost no blue staining on the rootsurface compared with wild-type plants, indicatinga lack of APase activity. This result is consistentwith that previously reported for the pho3 mutant,which contains a point mutation in the SUC2 gene(Zakhleniuk et al., 2001). Expression of four PSI genes(AtPT2, ACP5, RNS1, and AtIPS1) was also analyzed.Induction of these genes in roots of suc2-5 plants wassignificantly reduced, especially for AtIPS1 (Fig. 8C).However, in shoot tissues, the expression of these PSIgenes, except RNS1, was higher than that of the wildtype (Fig. 8D). Similar result was obtained when thesuc2-5 mutation was introduced into the AtPT2-GUSmarker line through a genetic cross (i.e. GUS expres-sion under Pi starvation was enhanced in shoot tissueand blocked in root tissue; Fig. 8B). These contrastingexpression patterns of PSI genes could be correlatedwith altered accumulation patterns of Suc in suc2-5plants. In an early study, Gottwald et al. (2000) hadshown that the transport of Suc from shoot to rootthrough the phloem is greatly blocked in suc2mutants;thus, more Suc is accumulated in shoots and less inroots. These results strongly supported the notion thatthe level of Suc inside plant cells is a major determi-nant of plant responses to Pi starvation.

Suc Plays a Global Regulatory Role in Mediating Plant

Responses to Pi Starvation

To determine the effects of the hps1 mutation (or thehigh accumulation of Suc) at the genomic level, wecompared the gene expression profiles between hps1and wild-type plants by microarray analysis. ThemRNAs used for analysis were isolated from 9-d-oldseedlings (the same batch of mRNAs used for real-time PCR analysis as shown in Fig. 2). All the com-parisons were made against the gene expression levelof wild-type plants on P+ medium. The corrected Pvalue (P , 0.001) was used to select the genes forcomparison, and only those genes whose expressionwas 2-fold higher or lower compared with that ofwild-type plants on P+ medium were used for anal-ysis. The overall changes in gene expression patternsare shown in Supplemental Figure S9. This study ismainly focused on the analysis of genes whose ex-pression is up-regulated.

We first compared the gene expression profiles ofwild-type plants under P+ and P2 conditions. Underour experimental conditions, expression of 325 geneswas induced, and 133 genes were repressed whenwild-type plants were grown under Pi starvation.Among these 325 induced genes were those eight PSIgenes used as markers in real-time PCR analysis (Fig.

Figure 5. Comparisons of root morphologies between wild-type andhps1 seedlings. Wild-type (WT) and hps1 plants were grown under P+and P2 conditions for 9 d on vertically oriented petri plates. A,Photographs are of representative wild-type and hps1 whole seedlingsgrown on MS P+ and P2 media. B and C, Microscopic images of rootmiddle parts (B) and root tips (C) with intact root hair from plants grownon MS P+ and P2 media.

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2; Table I; Supplemental Table S2). Pi starvation-in-duced genes included key Pi signaling componentssuch as AtIPS1, At4, and a SPX domain-containingprotein that play important roles in regulating Pihomeostasis (Burleigh and Harrison, 1999; Martın

et al., 2000; Franco-Zorrilla et al., 2007). The 1,2-diac-ylglycerol 3-b-galactosyltransferase genes thought tobe involved in the synthesis of galactolipid were alsostrongly induced. Galactolipids are shown to replacephospholipids in biomembranes under Pi starvation(Dormann and Benning, 2002). Another interestingobservation is that 15 peroxidase genes are highlyinduced by Pi starvation (Supplemental Table S2).These may be involved in scavenging ROS accumu-lated during Pi deficiency.

When comparing the gene expression profiles be-tween wild-type and hps1 plants under normal growthconditions, we found that in hps1 mutants, there are1,608 genes induced (Supplemental Table S1) and1,071 genes repressed. Among the 1,608 induced genesin hps1 plants, 238 genes could also be induced by Pistarvation in wild-type plants (Supplemental Fig. S9;Supplemental Table S2). In other words, elevated Suclevel alone was sufficient to induce 73% (238 out of325) of PSI genes observed in wild-type plants. Table Ilists some PSI genes induced in hps1 plants whengrown in Pi-sufficient medium. AtIPS1, At4, and SPX3are important regulators for Pi homeostasis, and theywere among the highly induced genes in hps1 plants(Table I), suggesting that these signaling componentsact downstream of Suc in regulating plant responses toPi starvation. Besides those genes, others that aredirectly involved in Pi transport (AtPT1 and AtPT2),mobilization (ACP5 and RNS1), and synthesis ofgalactolipids (1,2-diacylglycerol 3-b-galactosyltrans-ferase) were also induced in hps1 plants (Table I;Supplemental Table S1). The induction of these PSIgenes by elevated Suc level could be expected througha Suc-specific signaling pathway or by altered carbonmetabolism. In either case, Suc seems to act at a veryearly step to trigger PSI gene expression.

We also compared the gene expression patterns ofwild-type and hps1 plants on P2 medium. In contrastto wild-type plants on P2 medium, in which only 328genes were induced, there were 3,678 genes induced inhps1 plants (Supplemental Fig. S9). Among these 3,678genes, 305 genes overlapped with 328 genes inducedin wild-type plants under Pi deficiency. Interestingly,57% of the 328 PSI genes induced in wild-type plantswere hyperinduced in hps1 plants (Table I; Supple-mental Table S3). For example, on P2 medium, a hy-pothetical protein genewas induced 167-fold in thewildtype but 2,706-fold in hps1; similarly, an APase gene(PAP25; At4g36350) was induced 101-fold in wild-typeplants but 2,596-fold in hps1. AtIPS1, At4, and SPXdomain-containing protein were also among thesehyperinduced genes. In fact, AtIPS1 was the highestinduced gene in hsp1 plants on P2 medium. Theinduction of the 1,2-diacylglycerol 3-b-galactosyltrans-ferase gene had increased significantly in hps1 plantscompared with the wild type (230.9-fold versus 20.5-fold), suggesting that an active synthesis of galactoli-pids might take place in hps1 plants. These resultsextended our observations on the effects of hps1 muta-tion (or high Suc) on the expression of PSI genes (Fig. 2).

Figure 6. Accumulation of Suc in the hps1 mutant due to overexpres-sion of the SUC2 gene. A, Diagram showing the T-DNA insertion siterelative to SUC2 in hps1 and suc2-5 mutants. Untranslated regions areshown in gray, exons in black, and introns as thick lines. Bar, Basta-resistant gene; LB, T-DNA left border; RB, T-DNA right border. B,Relative expression levels of SUC2 in 9-d-old wild-type (WT) and hps1seedlings. C, Suc uptake rates of wild-type and hps1 plants. Wild-typeand hps1 plants were grown on MS medium for 3 weeks, and the rootswere immersed in the uptake solution containing [14C]Suc and incu-bated for 2 h. The amount of [14C]Suc uptake was determined by ascintillation counter. FW, Fresh weight. D, Soluble sugars wereextracted from 9-d-old wild-type and hps1 seedlings grown on MSP+ and MS P2 media, and Suc contents were determined by gaschromatography-mass spectrometry. White bars, Wild-type plants;black bars, hps1 plants. Asterisks indicate significant differences com-pared with the wild type (P , 0.05, t test).





The strong enhancing effect of high Suc on PSI geneexpression may come from the interaction betweenSuc and Pi. In fact, we found that there existed bothsynergistic and antagonistic interactions between Sucand Pi starvation signals. For example, the AtIPS1gene was induced to 80-fold in hps1 plants on P+medium and 167-fold in wild-type plants on P2medium; however, in hps1 plants grown on P2 me-dium, this gene was induced by 2,706-fold (Table I).Similarly, a purple acid phosphatase (PAP25) gene wasinduced to 101- and 13-fold, respectively, by Pi star-vation and hps1 mutation (or high Suc), but when hsp1was Pi starved, the induction reached 2,596-fold (TableI). In contrast, a putative peroxidase gene (At1g49570)was induced 19- and 46-fold by Suc and Pi starvationindividually, but when both treatments were com-bined, the induction was only 14-fold, thus suggestingan antagonistic interaction. In summary, we havefound 78 genes whose expression was synergisticallyinduced by hps1 mutation and Pi starvation. AndSuc and low Pi together had antagonistic effects onthe expression of 60 genes (Supplemental Table S4).These results indicated that elevated levels of Suc hadbroader implications on Pi starvation-induced geneexpression in plants.

DISCUSSION

When plants are subjected to Pi starvation, theydisplay numerous adaptations to cope with the stress.There is growing evidence that Suc plays an importantrole in PSI responses (Zakhleniuk et al., 2001; Liu et al.,2005; Karthikeyan et al., 2007). Several reports, in-cluding this work, have shown that Pi starvation ledto increased accumulation of Suc in plant leaves(Hammond and White, 2008). Increased Suc levels inleaves of Pi-deficient plants and activation of PSI geneshave provided a tangible link between Suc and Pi (Liuet al., 2005; Karthikeyan et al., 2007). This work definesa broader role for Suc in many of the observed Pistarvation responses in Arabidopsis and tries to ex-plore how Suc acts at the molecular level in regulatingthese responses through genetic and genomic analysesof a Suc overaccumulation mutant, hps1.

hps1 was identified as an Arabidopsis mutant thatexhibited hyperinduction of PSI marker genes underPi-deficient conditions. The products of these PSImarker genes are thought to be directly involved inPi transport (AtPT1, AtPT2, Pht1;5), mobilization(ACP5 and RNS1), and control of Pi distribution be-tween shoots and roots (At4, AtIPS1, and miRNA399),

Figure 7. Comparison of AtPT2-LUC expressionand plant morphology of wild-type, hps1, andtransgenic lines overexpressing various SUCgenes. A, Nine-day-old seedlings of wild-type(WT), hps1, and two transgenic lines expressing35S-SUC2 grown on MS P+ (left panels) and P2(right panels) media were sprayed with luciferins,and the imageswere takenwith a CCD camera. B,Nine-day-old seedlings of wild-type, hps1, andtransgenic lines overexpressing various SUCgenes were sprayed with luciferins, and the im-ages were taken with a CCD camera. Top panelsshow LUC activity, and bottom panels show plantmorphology.

Lei et al.




which are critical for plants to maintain Pi homeostasisunder Pi deficiency conditions. Further comparison ofexpression profiles between wild-type and hps1 plantsat the genomic scale revealed that, among 325 Pistarvation-induced genes found in the wild type, theinduction of 190 genes (about 60%) was greatly en-hanced in hps1 plants (Supplemental Table S3). Theseresults indicated that mutation of the HPS1 gene has aglobal impact on PSI gene expression. Molecular andgenetic analyses indicated that the mutant phenotype

is caused by the overexpression of the SUC2 gene,which resulted in high accumulation of Suc in bothshoot and root tissues by enhanced Suc uptake fromthemedium. This mutant offered genetic evidence thatSuc is an important component for regulating PSI geneexpression. Previous studies have shown that changein photosynthate level had a strong effect on PSI geneexpression in white lupin and Arabidopsis (Liu et al.,2005, 2010b; Karthikeyan et al., 2007). This was dem-onstrated through stem girdling and photoperiod ma-

Figure 8. Comparison of APase activity and PSI gene expression in wild-type and suc2-5 mutant plants. Wild-type and suc2-5seedlings were grown on MS P+ medium with Suc for 5 d and then transferred to MS P2medium with no added Suc for another5 d. A, The APase activities on root surfaces of wild-type (WT) and suc2-5 seedlings grown on MS P2medium as shown by BCIPstaining. B, Histochemical analysis of AtPT2-GUS expression in wild-type and suc2-5 seedlings grown on MS P2 medium. C,Quantitative analysis of expression of four PSI genes in roots of wild-type and suc2-5 seedlings grown on MS P+ and MS P2media. D, Quantitative analysis of expression of four PSI genes in shoots of wild-type and suc2-5 seedlings grown on MS P+ andMS P2 media. The PSI genes examined in C and D encode a high-affinity Pi transporter (AtPT2), an APase (ACP5), an RNase(RNS1), and a noncodingmRNA (AtIPS1). Asterisks indicate significant differences comparedwith the wild type (P, 0.05, t test).White bars, wild-type plants; black bars, suc2-5 plants.





nipulation. However, in these experiments, it was notpossible to separate the role of Suc from other trans-locatable components acting as potential signalingmolecules. In this work, the wild-type and hps1 plantswere maintained at regular growth conditions (i.e. 16-h-light/8-h-dark photoperiod with 100 mmol m22 s21

fluorescent light). Thus, this study provided morecompelling evidence for the causal relationship be-tween the elevated Suc level and enhanced PSI geneexpression. Analyses of a SUC2 null allele, suc2-5,further support the notion that Suc is a major deter-minant of PSI gene expression. In the Arabidopsisgenome, there are nine annotated Suc transportergenes (Sauer, 2007). However, SUC2 is the only Suctransporter that is specifically involved in phloemloading of Suc from leaf mesophyll cells (Sauer et al.,2004). It has been shown that the disruption of SUC2function led to high accumulation of Suc in leaves andblocked Suc transport from leaves to root tissues(Gottwald et al., 2000). As a consequence, when suc2-5is grown on Suc-free P2 medium, the expression offour PSI marker genes is enhanced in shoots. Incontrast, the expression of these PSI genes and theinduction of APase were largely attenuated in roots.Transfer of Suc from shoot to root seems to be a crucialpart of signaling during Pi starvation, and Suc trans-porters have an important role in regulating sugarmovement. Furthermore, the ectopic overexpressionof SUC1 and SUC5, which have similar Suc transportproperties as SUC2, can also recapitulate the hps1mutant phenotype in wild-type plants (Fig. 7; i.e.enhancing plant sensitivity to Pi starvation), demon-strating that it is the level of Suc, rather than a

particular Suc transporter, that affects the magnitudeof plant responses to Pi starvation.

In this work, we noticed that the expression levels ofseven out of eight PSI genes examined by real-timePCR were largely enhanced in the hps1 mutant com-pared with the wild type even when they were grownunder Pi-sufficient conditions (Fig. 2). The enhancingeffect on the expression of some PSI genes (AtPT1,AtPT2, LePT1, LaSAP1, At4, and RNase2) by exoge-nously applied Suc was also reported by other groupsin white lupin and Arabidopsis (Lejay et al., 2003;Karthikeyan et al., 2007). These results indicated thatincreased Suc level alone is sufficient to induce PSIgene expression independent of low-Pi stress. Further-more, our genomic studies indicated that 73% (238 outof 325) of Pi starvation-induced genes can be directlyinduced by elevated levels of Suc in hps1 plants. Thesegenes include some of the known regulatory compo-nents involved in Pi starvation responses and manydownstream target genes that directly participate in Pitransport and mobilization. Although the elevated Suclevel is sufficient to induce or enhance the expressionof many PSI genes in Pi-sufficient conditions, thedegree of its induction is much lower compared withthat induced by Pi starvation. This observation im-plied that Suc is not just a simple link between low-Pisignal and PSI gene expression; it needs to interactwith other factors under Pi starvation conditions toorchestrate the expression of a battery of PSI genes.This notion is further supported by the genomic datathat 78 genes are synergistically induced by both Pistarvation and high levels of Suc, and the expression of60 genes is antagonistically regulated (Table I; data not

Table I. The top 20 genes that are synergistically induced by hps1 mutation and Pi starvation

Data are based on microarray analysis. Values are means of three biological replicates where the fold changes are normalized to signal levels inthe wild type on P+ medium.

Arabidopsis Genome

Initiative CodeDescription

Fold Change

hps1, P+ Wild Type, P2 hps1, P2

AT3G09922 AtIPS1 74.9 167.6 2,706.5AT4G36350 Purple acid phosphatase 25 13.9 101.4 2,596.2AT1G73220 Putative sugar transporter 11.2 183.0 783.3AT2G45130 SPX3 20.1 42.9 594.9AT2G24000 Ser carboxypeptidase S10 family protein 8.0 9.4 287.5AT4G34210 E3 ligase SCF complex subunit SKP1/ASK1 14.1 11 244.6AT2G11810 1,2-Diacyglycerol 3-b-galactosyltransferase 25.9 20.5 230.9AT5G22570 WRKY38 transcription factor 21.3 20.3 200.3AT3G03530 Phosphoesterase family protein 6.6 18.9 197.4AT4G24890 Purple acid phosphatase 24 4.9 9.1 121.7AT5G13320 Auxin-responsive GH3 family protein 12.4 10.2 115.6AT2G34210 KOW domain-containing transcription factor 3.9 11.3 65.6AT5G64000 3#(2’),5#-Bisphosphate nucleotidase 4.2 5.2 53.7AT5G03545 At4 11.0 14.0 48.6AT3G05630 PLDz2 7.0 6.9 47.6AT1G21320 RNA recognition motif-containing protein 3.0 6.6 39.9AT3G04530 Phosphoenolpyruvate carboxylase kinase 2 2.7 4.1 35AT3G52820 Purple acid phosphatase 22 2.6 6.1 33.4AT2G38940 AtPT2 3.1 6.3 27.1AT1G49570 Peroxidase 45.6 18.7 13.6

Lei et al.




shown). Interestingly, the expression of AtIPS1, a keyregulator of Pi homeostasis, is highest among thegenes induced in Pi-starved hps1 plants. Expressionof this gene is also synergistically induced by thecombined effects of Suc and low-Pi signals (Table I).These synergistically induced genes could be the con-verging points for Suc and Pi signals to regulatemultiple plant responses to Pi starvation. The syner-gistic interaction of Suc and low-Pi signals in regulat-ing gene expression at the genomic level has also beenobserved in a previous study using exogenously ap-plied Suc to Pi-starved excised leaves (Muller et al.,2007). Thus, Suc appears to act as a major regulator ofthe expression of genes that are involved in plantresponses to Pi starvation.Suc also plays an important role in Pi starvation-

induced lateral root formation and root hair develop-ment (Jain et al., 2007). Increased production of lateralroots and root hairs and inhibition of primary growthare the major morphological changes associated withPi starvation, which are thought to enhance Pi acqui-sition efficiency. When Arabidopsis seedlings weregrown on Suc-free Pi-deficient medium, the develop-ment of lateral roots and root hairs was greatly sup-pressed (Nacry et al., 2005; Jain et al., 2007;Karthikeyan et al., 2007), suggesting that Suc is re-quired for such Pi starvation-induced morphologicalchanges. Further experimental evidence showed thatthe role of Suc might be to enhance plant sensitivity toauxin, an important mediator of lateral root develop-ment (Casimiro et al., 2001; Jain et al., 2007). Using thehps1 mutant, we were able to show that elevated Suclevel alone is sufficient to inhibit primary root growthand enhance lateral root formation and root hairdevelopment, even when plants are grown under Pisufficiency (Fig. 5), a phenotype that mimics a consti-tutive root Pi starvation response. Besides regulatingPSI gene expression and root architecture changes, thisstudy also shows that Suc level has a great effect onmultiple Pi starvation responses, including the pro-duction of APase and the accumulation of starch andanthocyanin. In hps1 mutants that accumulate highlevels of Suc, the production of APases, starch, andanthocyanin is greatly enhanced even in P+ medium,which mimics the expression of PSI genes and rootdevelopment. In contrast, the APase activity on theroot surface is largely reduced in the suc2-5 mutant.The high accumulation of anthocyanin and starch inshoots, and the reduced production of APase in roots,were also observed in pho3, another mutant allele ofthe SUC2 gene (Zakhleniuk et al., 2001; Lloyd andZakhleniuk, 2004). Taken together, these results showthat the hps1 mutant has enhanced sensitivity in mul-tiple plant responses to Pi starvation, and this is pri-marily due to increased levels of Suc, which acts as aglobal regulator of plant responses to Pi starvation.It is becoming evident that when plants are under Pi

starvation, Suc level increases in their leaf tissues,probably because of the changes of gene expression orenzymatic activities that are associated with carbohy-

drate metabolism. In Arabidopsis, Suc is transportedfrom leaf to root due to the action of SUC2. Thetransported Suc then acts as a signal to trigger the rootPi starvation responses in addition to meeting thenutrient demands of an expanding root system. Ourresults also demonstrated that the plant Pi starvationresponse triggered by Suc is not due to the sequestra-tion of cellular Pi by elevated sugar level, since the Piconcentration in root tissues of hps1 plants is actuallyhigher than that of wild-type plants under both Pi-sufficient and Pi-deficient conditions (Fig. 4). Since Sucwill be broken down to Glc and Fru when it enters themetabolic pathway, it could be argued that it may notbe the real signaling molecule for eliciting the plant Pistarvation response. This study showed that the levelsof Glc and Fru in hps1 plants are lower than in wild-type plants (Supplemental Fig. S6), excluding thepossibility that enhanced plant Pi sensitivity in hps1is caused by an increased level of Glc or Fru. Theseresults support the hypothesis that a Suc-specific sig-naling pathway exists in triggering plant responses toPi starvation.

In summary, our work demonstrates that Suc is aglobal regulator of plant responses to Pi starvation.Through genomic analyses, we showed that elevatedlevels of Suc can directly alter the expression of a largenumber of PSI genes that are involved in Pi signaling,transport, mobilization, and allocation between shootsand roots, which will ultimately help plants maintainPi homeostasis and better adapt to this nutritionalstress. A challenging task ahead is to identify signalingcomponents that are the direct targets of Suc andunderstand how this carbohydrate signal is perceivedand transmitted to elicit plant Pi responses at themolecular level. The data generated from our compar-ative genomic study will provide a valuable resourceto search for these Suc-regulated signaling compo-nents. Alternatively, screening for genetic suppressorsof hps1 and cloning the corresponding genes may alsobe an effective way to achieve this goal.

MATERIALS AND METHODS

Plant Materials and Growth Medium

All Arabidopsis (Arabidopsis thaliana) plants used in this study, including

mutants and transgenic plants, were in the Columbia background. Seeds

were surface sterilized in 20% (v/v) household bleach solutions and washed

with distilled water three times. Then they were planted on 8-cm-diameter

sterile plates containing full-strength medium (MS P+) supplemented with

1% Suc and 1.2% agar or MS P2 medium (substituting 0.6 mM K2SO4 for

1.2 mM KH2PO4). After being cold stratified at 4�C for 2 d, these plates were

placed vertically in the growth room. The growth room conditions were

24�C, 16-h-light/8-h-dark photoperiod, with 100 mmol m22 s21 fluorescent

light.

Isolation of Mutants

T2 seeds were plated directly on MS P2 medium plates. After 8 d in the

growth room, 100 mM luciferin in 0.1% Triton X-100 was uniformly sprayed on

the seedling surface, and fluorescence images were captured by exposing the

seedlings for 5 min on a highly sensitive iXon CCD camera (Andor Technol-





ogy). Seedlings with altered LUC expression compared with wild-type plants

were selected as candidates and transferred to soil. Seeds harvested from the

putative mutants were retested for inheritance of the observed phenotype.

The hps1 mutant presented in this work was backcrossed to wild-type plants

before further characterization.

Analysis of APase Activity

In vivo APase assay was performed on 9-d-old seedlings vertically grown

on P+ or P2 medium as described by Zakhleniuk et al. (2001). Agar (0.5%,

w/v) containing 0.01% (w/v) BCIP was evenly overlaid on the root surface.

After 8 h of color development, photographs were taken with a camera

attached to a stereomicroscope (Olympus SZ61).

For in-gel APase assay, about 50 mg of seedlings was ground in liquid N2

and total protein was extracted using extraction buffer (0.1 M KAc, 20 mM

CaCl2, 2 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride). The same

amount of protein was separated on a 10% SDS-PAGE gel. After electropho-

resis, the gel was washed in distilled, deionized water at 4�C six times (10 min

each) to renature the proteins and equilibrated twice with sodium acetate

buffer (50 mM sodium acetate and 10 mM MgCl2, pH 4.9). Then, the gel was

stained with 0.5 mg mL21 Fast Black K coupled with 0.3 mg mL21 b-naphthyl

phosphate dissolved in sodium acetate buffer at 37�C for 4 h (Trull and

Deikman, 1998; Zakhleniuk et al., 2001).

Quantitative analysis of APase activity was done by mixing 10 mL of

protein extract with 470 mL of MES buffer (15 mM MES, pH 5.5, and 0.5 mM

CaCl2) and 20 mL of p-nitrophenyl phosphate, disodium salt. After incuba-

tion at 27�C for 30 min, the reaction was terminated with 500 mL of 0.25 M

NaOH. The developed color was determined spectrophotometrically at 412

nm, and APase activity was expressed as A412 mg21 protein (Richardson

et al., 2001).

Quantitative Measurements of Anthocyanin,Chlorophyll, and Inorganic Pi

Anthocyanin was extracted with 1% HCl in methanol from shoots of the

seedlings grown on P+ or P2 medium for 9 d. Anthocyanin content was

measured as described previously (Mita et al., 1997). Chlorophyll was

extracted with 80% acetone from leaves of the seedlings grown on P+ or P2medium for 9 d. The chlorophyll a, chlorophyll b, and carotene contents were

measured as described previously (Arnon, 1949). Inorganic Pi was determined

using the method described by Ames (1966).

Starch Analysis

For starch analysis, 11-d-old seedlings were immersed in 96% (v/v)

ethanol overnight to remove pigments and stained with 1% iodine for 30

min. After a brief wash in distilled, deionized water, the samples were kept in

96% ethanol, and starch accumulation was observed with a stereomicroscope.

Histochemical Analysis of GUS Activity

For histochemical analysis of GUS activity, whole seedlings were in-

cubated for 6 h at 37�C in GUS staining solution (2 mM 5-bromo-4-chloro-

3-indolyl-b-glucuronic acid in 50 mM sodium Pi buffer, pH 7.2) containing

0.1% Triton X-100, 2 mM K4Fe(CN)6, 2 mM K3Fe(CN)6, and 10 mM EDTA.

The stained seedlings were then transferred to 50%, 70%, and 100% (v/v)

ethanol to remove the chlorophyll. The whole seedlings were photographed

with a stereomicroscope (Olympus). The images of the shoot-root junctions

and root tips were taken with a differential interference contrast microscope

(Olympus).

Inductively Coupled Plasma-Optical EmissionSpectrometry Elemental Profiling

Nine-day-old seedlings were cut into shoot and root parts and dried at

80�C for 2 d. The tissuewas digested for 8min at 120�C and for 15min at 160�Cwith concentrated HNO3 and diluted with distilled, deionized water. The

contents of phosphorus, sodium, magnesium, potassium, calcium, zinc, iron,

manganese, and sulfur were determined by inductively coupled plasma-

optical emission spectrometry (ICP-OES; IRIS Intrepid II XSP; Thermo Electron).

Cloning of T-DNA-Flanking Sequence in hps1 and

Genotyping of suc2-5

The flanking sequence of the T-DNA insertion in the hps1 mutant was

cloned by the thermal asymmetric interlaced-PCR method (Liu et al., 1995).

The positive clones were sequenced and checked with T-DNA left border

primer (LB3, 5#-TGACCATCATACTCATTGCTG-3#) and gene-specific primer

(SUC2T, 5#-CTCGGATCGAAATTCGAAAG-3#).The suc2-5 T-DNA knockout line was obtained from the Arabidopsis

Biological Resource Center (SALK_087046) in the Columbia background. The

homozygous lines was confirmed using the T-DNA left border primer (LBb1.3,

5#-ATTTTGCCGATTTCGGAAC-3#) and gene-specific primers (SALK_087046R,

5#-TTTACCTGAGGGACGACACAATG-3#; and SALK_087046L, 5#-GTTTTTC-GGAGAAATCTTCGG-3#) as described (http://signal.salk.edu/tdnaprimers.2.

html).

Generation of Transgenic Plants

The genomic sequences of SUC1, SUC2, SUC4, and SUC5 were amplified

from wild-type Arabidopsis plants. The sequence of SUC3 was amplified by

RT-PCR from a wild-type plant. The PCR products were cloned into the vector

pEASY-Blunt (Transgen) and sequenced. Then, they were subcloned into the

plant T-DNA vector pCAMBIA1301 under the control of the cauliflower

mosaic virus 35S promoter. The vector has a hygromycin-resistant gene as a

selectable marker for plant transformation. These constructs were then

transformed into wild-type plants by the floral dip method (Clough and

Bent, 1998).

Quantitative Real-Time PCR Analysis

Total RNAwas extracted with the Qiagen RNAeasy kit. One microgram of

DNase-treated RNAwas reverse transcribed in a 20-mL reaction using reverse

transcriptase (Toyobo) according to the manufacturer’s manual. cDNA was

amplified using SYBR Premix Ex Taq (TaKaRa) on the Bio-Rad CFX96 real-

time PCR detection system. Ubiquitin-conjugating enzyme 21 mRNAwas used

as an internal control. The genes and their primers are listed in Supplemental

Table S5.

Quantitative Analysis of Sugar Contents

Nine-day-old seedlings were cut into shoot and root parts and ground in

liquid nitrogen thoroughly. Soluble sugar was extracted in 80% (v/v) ethanol

twice at 80�C for 15 min. Samples were centrifuged at 4,000 rpm for 10 min

and then dried at 85�C. For methoximation, 80 ml of methoxyamine hydro-

chloride in pyridine (20mgmL21) was added at 30�C for 90 min. After, 80 ml of

N-methyl-N-trimethylsily-trifluoroacetamide was added, and the mixture

was incubated at 37�C for 30 min. The sugar derivatives were analyzed by

gas chromatography-mass spectrometry on an Agilent 7890A GC/5975 mass

spectrometer using a DB-5ms column (Agilent Technologies). A temperature

program was implemented as follows: initially at 180�C, followed by heating

to 300�C at 10�C min21, and then held at 300�C for a further 10 min.

Myoinositol was used as an internal standard.

Suc Uptake

Three-week-old seedlings grown on MS medium without supplemented

Suc were used for Suc uptake experiments. After incubation in pretreatment

solution (MS liquid medium, pH 5.7) for 30 min, the roots were incubated in

the uptake solution (MS medium with 0.1% Suc, pH 5.7) containing [14C]Suc

(0.5 mCi mL21) and incubated for 2 h. After two washes in desorption solution

(MS liquid medium and 1% Suc, pH 5.7), the seedlings were placed in

scintillation vials and 3 mL of scintillation cocktail was added. The amount of

[14C]Suc uptake was assessed on a scintillation counter and expressed as cpm

mg21 fresh weight.

Microarray Analysis

The microarray experiments were performed at ShanghaiBio Corporation

using the Agilent Arabidopsis microarray analysis platform. Total RNA was

isolated from three replicates of wild-type and hps1 seedlings grown on P+

and P2 media for 9 d using the RNase kit (Qiagen). First- and second-strand

Lei et al.




cDNA were generated using Moloney murine leukemia virus reverse tran-

scriptase with T7 oligo(dT)24 primer. Copy RNA was in vitro synthesized by

T7 RNA polymerase and labeled with Cyanine 3 (Agilent). After purification

and fragmentation, copy RNA was hybridized to the slides for 17 h at 65�Cusing the Agilent hybridization kit according to the manufacturer’s instruc-

tions. The gene probes on the chips are synthesized according to the cDNA

sequences released in The Arabidopsis Information Resource 8 (2008), The

Institute for Genome Research Plant Transcripts Assemblies (release 2; 2006),

RefSeq (release 30; 2008), and UniGene (build 67; 2008).

The Arabidopsis Genome Initiative locus numbers for the major genes

discussed in this article are as follows: SUC1 (AT1G71880), SUC2 (AT1G22710),

SUC3 (AT2G02860), SUC4 (AT1G09960), SUC5 (AT1G71890), At4 (At5G03545),

AtIPS1 (At3G09922),AtPT1 (At5G43350),AtPT2 (At2G38940),Pht1;5 (At2G32830),

RNS1 (At2G02990), miR399D (AT2G34202), and ACP5 (AT3G17790).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Map of the pSuperTag2 plasmid.

Supplemental Figure S2. Comparison of AtPT1-GUS expression between

wild-type and hps1 plants.

Supplemental Figure S3. APase activities in wild-type and hps1 plants.

Supplemental Figure S4. Comparison of ion contents between wild-type

and hps1 plants.

Supplemental Figure S5. Comparison of the number of lateral roots

produced in wild-type and hps1 mutant plants.

Supplemental Figure S6. Glc and Fru contents in wild-type and hps1

seedlings.

Supplemental Figure S7. AtPT2-LUC expression and morphologies of

wild-type and hps1 seedlings grown on MS Suc-free medium.

Supplemental Figure S8. Phenotypic and molecular analyses of the suc2-5

mutant.

Supplemental Figure S9. Diagrams showing the number of genes up- and

down-regulated by Pi starvation and hps1 mutation from microarray

analysis.

Supplemental Table S1. Genes whose expression is up- and down-

regulated in hps1 plants grown on P+ medium.


regulated in wild-type plants grown on P2 medium.


regulated in hps1 plants grown on P2 medium.


regulated by both Pi starvation and hps1 mutation.

Supplemental Table S5. Primers used for real-time PCR analysis.

ACKNOWLEDGMENTS

We thank Dr. Nigel Crawford for insightful discussion of the manuscript.

We also thank Ms. Ning Sui for technical help during this work and Dr. Zhi

Xing for assistance in ICP-OES analysis of element contents in plant tissues.

We are grateful to the Arabidopsis Biological Resource Center for providing

seed stock of the suc2-5 line (SALK_087046).

Received December 23, 2010; accepted February 20, 2011; published February

23, 2011.

LITERATURE CITED

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