Phosphate Deﬁciency Induces the Jasmonate Pathway and...genes (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007).Uponwounding,enhancedJA-Ilelevelspromote the degradation

Phosphate Deficiency Induces the Jasmonate Pathway andEnhances Resistance to Insect Herbivory1[OPEN]

Ghazanfar Abbas Khan, Evangelia Vogiatzaki, Gaétan Glauser, and Yves Poirier*

Department of Plant Molecular Biology, University of Lausanne, CH–1015 Lausanne, Switzerland (G.A.K.,E.V., Y.P.); and Neuchâtel Platform of Analytical Chemistry, University of Neuchâtel, CH–2009 Neuchâtel,Switzerland (G.G.)

ORCID IDs: 0000-0002-7557-6210 (G.A.K.); 0000-0003-2503-316X (E.V.); 0000-0001-8660-294X (Y.P.).

During their life cycle, plants are typically confronted by simultaneous biotic and abiotic stresses. Low inorganic phosphate (Pi)is one of the most common nutrient deficiencies limiting plant growth in natural and agricultural ecosystems, while insectherbivory accounts for major losses in plant productivity and impacts ecological and evolutionary changes in plant populations.Here, we report that plants experiencing Pi deficiency induce the jasmonic acid (JA) pathway and enhance their defense againstinsect herbivory. Pi-deficient Arabidopsis (Arabidopsis thaliana) showed enhanced synthesis of JA and the bioactive conjugate JA-isoleucine, as well as activation of the JA signaling pathway, in both shoots and roots of wild-type plants and in shoots of thePi-deficient mutant pho1. The kinetics of the induction of the JA signaling pathway by Pi deficiency was influenced byPHOSPHATE STARVATION RESPONSE1, the main transcription factor regulating the expression of Pi starvation-inducedgenes. Phenotypes of the pho1 mutant typically associated with Pi deficiency, such as high shoot anthocyanin levels and poorshoot growth, were significantly attenuated by blocking the JA biosynthesis or signaling pathway. Wounded pho1 leaveshyperaccumulated JA/JA-isoleucine in comparison with the wild type. The pho1 mutant also showed an increased resistanceagainst the generalist herbivore Spodoptera littoralis that was attenuated in JA biosynthesis and signaling mutants. Pi deficiencyalso triggered increased resistance to S. littoralis in wild-type Arabidopsis as well as tomato (Solanum lycopersicum) and Nicotianabenthamiana, revealing that the link between Pi deficiency and enhanced herbivory resistance is conserved in a diversity of plants,including crops.

While a large number of studies have examined theadaptation and signaling pathways involved in the re-sponse of plants to a single stress, plants grown in na-ture typically are confronted by simultaneous stresses,both biotic and abiotic. The application of combinedstresses often leads to responses that are distinct fromthose to individual stresses and display deleteriousinteractions (i.e. the adaptation to one stress leads toenhanced sensitivity to a distinct stress; Mittler andBlumwald, 2010). This is exemplified by the an-tagonistic interactions between the salicylate andjasmonic acid (JA)/ethylene pathways involved in theresponse to biotrophic pathogens and herbivory, re-spectively (Spoel et al., 2003). However, few examples of

synergistic interaction between abiotic and bioticstresses have been described, such as the positive effectof short-term potassium deficiency on resistance tothrips (Frankliniella spp.; Armengaud et al., 2010).

The essential element phosphorus is one of the mostlimiting nutrients restraining plant growth in bothnatural and agricultural ecosystems (Lynch, 2011;MacDonald et al., 2011). Plants essentially acquirephosphorus through the uptake of soluble inorganicphosphate (Pi) by the roots via the PHT1 Pi transporters(Poirier and Jung, 2015). Although the phosphorus levelin soil may be high, the concentration of soluble Pi inmost soils is kept very low (low micromolar range) dueto the formation of insoluble complexes of Pi with cal-cium, iron, and aluminum and the assimilation of Pi bythe soil microflora. High-yield crop production in amajority of agricultural land is thus dependent on fer-tilizers based on nonrenewable phosphorus-rich mines(Obersteiner et al., 2013).

Plants respond to Pi-deficient conditions throughnumerous changes at the biochemical, developmental,and gene expression levels (Chiou and Lin, 2011;Plaxton and Tran, 2011; Zhang et al., 2014). These in-clude reductions in shoot growth and primary rootelongation, enhancement of the growth of lateral rootsand root hairs, replacement of phospholipids by sulfo-lipids and galactolipids, secretion of phosphatases toscavenge Pi from organic sources, and accumulationof anthocyanins. At the molecular level, PHOSPHATE

1 This work was supported by the Swiss National Foundation(grant nos. 31003A–138339 and 31003A–159998).

* Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Yves Poirier ([email protected]).

G.A.K. and Y.P. designed experiments and wrote the article, andY.P. supervised the whole project; G.A.K. performed all experiments,except grafting experiments, which were performed by E.V.; G.G.provided technical and analytical support for jasmonic acid measure-ments.

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632 Plant Physiology�, May 2016, Vol. 171, pp. 632–644, www.plantphysiol.org � 2016 American Society of Plant Biologists. All Rights Reserved.

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STARVATION RESPONSE1 (PHR1) is the main tran-scription factor responsible for the majority of tran-scriptional responses triggered by Pi deficiency (Rubioet al., 2001; Bustos et al., 2010). PHR1 controls theexpression of miR399, a microRNA that moves fromshoots to roots to repress the PHO2 transcript (Lin et al.,2008; Pant et al., 2009). PHO2 encodes a ubiquitin ligaseinvolved in the degradation of PHO1, a Pi exporterexpressed in the root xylem parenchyma and involvedin the transfer of Pi from roots to shoots (Hamburgeret al., 2002; Liu et al., 2012).Herbivory is a major biotic stress responsible for the

loss of a substantial fraction of the biomass producedannually in terrestrial ecosystems (Cyr and Face, 1993).JA is one of the most important hormones involved inthe response of plants to herbivory-induced wounding,controlling the majority of insect-regulated genes inArabidopsis (Arabidopsis thaliana) leaves (Acosta andFarmer, 2010). The synthesis and accumulation of JAand of its conjugate jasmonic acid-isoleucine (JA-Ile) areincreased within minutes after wounding (Glauseret al., 2008). The JA signaling pathway involves the JAZproteins acting as repressors of the MYC2 transcriptionfactor, promoting the transcription of JA-responsivegenes (Chini et al., 2007; Thines et al., 2007; Yan et al.,2007). Uponwounding, enhanced JA-Ile levels promotethe degradation of JAZ proteins, including JAZ10, viaJA-Ile interaction with the COI1 protein, an F-box pro-tein recruiting a SCF-type E3 ubiquitin ligase (Yan et al.,2009). JAZ10 andMYC2 form a negative feedback loop,with JAZ10 degradation triggered by JA/JA-Ile, lead-ing to its transcriptional activation via MYC2 (Chiniet al., 2007; Thines et al., 2007; Yan et al., 2007).JA induction and Pi starvation share some common

phenotypes, including growth reduction and anthocy-anin accumulation, suggesting a potential role of JA inthe Pi deficiency response (Shan et al., 2009; Yang et al.,2012). Although numerous transcriptomic studies ex-amining the responses of leaves or whole roots to Pideficiency have been performed in model plants, suchas Arabidopsis (Hammond et al., 2003; Misson et al.,2005; Morcuende et al., 2007; Müller et al., 2007; Bustoset al., 2010; Woo et al., 2012) and rice (Oryza sativa;Wasaki et al., 2006; Li et al., 2010; Secco et al., 2013), onlyone study (Morcuende et al., 2007) described changes inthe expression of JA biosynthetic and signaling genes. Atranscriptomic study focusing on Arabidopsis root tipsrevealed the down-expression of several JA-regulatedgenes in the low phosphorus insensitive4 (lpi4) mutant,indicating a potential role of JA in primary root elon-gation under Pi-deficient conditions (Chacón-Lópezet al., 2011). Changes (up- and down-regulation) in theexpression of some genes involved in the JA pathwayby Pi deficiency also have been described in sometranscriptomic studies in legumes, such as common bean(Phaseolus vulgaris; Aparicio-Fabre et al., 2013) and whitelupin (Lupinus albus; O’Rourke et al., 2013; Wang et al.,2014). However, the potential role and consequencesof the induction of the JA pathway in the response toPi deficiency remain unknown.

In this work, we show that the JA signaling pathwayand the synthesis of JA and JA-Ile are induced by Pideficiency in both roots and shoots of Arabidopsis.Activation of the JA pathway in the Pi-deficient mutantpho1 leads to a reduction in shoot growth, the accu-mulation of anthocyanins in leaves, the hyperactivationof the wound response, and an increased resistance toSpodoptera littoralis herbivory. Pi deficiency also in-creased the resistance of Nicotiana benthamiana and to-mato (Solanum lycopersicum) to S. littoralis, indicatingthat Pi deficiency-mediated herbivory resistance isconserved across several plant species.

RESULTS

Pi Deficiency Induces the JA Biosynthetic andSignaling Pathways

We first analyzed the potential effects of Pi deficiencyon the JA pathway by measuring the expression ofJAZ10, a gene participating in the JAZ-COI1-MYC2signaling core and a marker for the activation of theJA signaling pathway (Chini et al., 2007; Thines et al.,2007; Yan et al., 2007). JAZ10 expression was increased4- to 5-fold in both shoots and roots of wild-typeColumbia-0 (Col-0) Arabidopsis plants grown on me-dium containing low Pi (100mM) comparedwith high Pi(1 mM; Fig. 1, A and B). Under the same conditions, theexpression of JAZ10 was highly induced in the tips(17-fold) of roots grown on low-Pi medium (Fig. 1C).The expression of JAZ10 in both shoots and roots ofplants grown in high or low Pi was strongly reduced inthe JA biosynthetic mutant aos lacking allene oxidaseactivity (Park et al., 2002) and the JA signaling mutantcoi1-34 (a fertile allele of coi1; Acosta et al., 2013; Fig. 1,A and B). The expression pattern of two other markersof the JA pathway in leaves, namely VSP2 and LOX2,mirrored the expression of JAZ10 in leaves, being in-duced in leaves of Pi-deficient plants and this inductionbeing abrogated in the aos and coi1-34 mutants (Fig. 1,D and G). In roots, VSP2 expression was induced in theroot tips of Pi-deficient plants but no increase wasdetected when whole roots were analyzed, indicatingthat VSP2 expression in Pi-deficient plants is mostlikely restricted to the root tip (Fig. 1, E and F). LOX2expression in roots was not affected by Pi deficiencyin either whole roots or root tips (Fig. 1, H and I). Thisis in agreement with previous studies showing that, incontrast to leaves, LOX2 expression remains very low inroots of plants with induced JA biosynthesis followingwounding (Grebner et al., 2013; Gasperini et al., 2015).

The expression pattern of JA-responsive genes inplants grown on low-Pi medium and their dependenceon both AOS and COI1 suggested that JA biosynthesiswas induced under these conditions. Thus, JA and JA-Ile were quantified using liquid chromatography-massspectrometry. Both JA and JA-Ile levels increased inshoots (Fig. 2,A andB) and roots (Fig. 2, E and F) ofCol-0plants grown in low-Pi medium. Wounding of leaves

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Figure 1. Genes involved in JA biosynthesis and signaling are induced in shoots and roots of Pi-deficient plants. A to I, Expressionlevels of JAZ10 (A–C), VSP2 (D–F), and LOX2 (G–I) measured in shoots (A, D, and G), whole roots (B, E, and H), and root tips(3 mm; C, F, and I) in Col-0 or the aos and coi1-34mutants. Plants were grown for 12 d in medium containing 1mM (black bars) or100 mM (gray bars) Pi. J, Expression levels of JAZ10, VSP2, and LOX2measured in shoots of Col-0 (black bars) and pho1-7 (gray

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resulted in a greater accumulation of JA and JA-Ilecompared with Pi deficiency, and the combination ofboth wounding and Pi deficiency resulted in small butsignificant increases of JA and JA-Ile compared withwounding alone (Fig. 2, C and D).The PHO1 gene is involved in the loading of Pi into

the root xylem for its transfer to shoots; consequently,pho1 mutants are Pi deficient in shoots (Hamburgeret al., 2002). Shoots of pho1 mutants show the typicalphenotypes related to Pi deficiency, including reducedgrowth, anthocyanin accumulation, a replacement of

phospholipids with galactolipids and sulfolipids, and in-duction of Pi deficiency-related genes (Hamburger et al.,2002; Rouached et al., 2011). The pho1 mutant is thus anexcellent tool to investigate shoot Pi deficiency and itslong-term effects in soil-grown plants. Two alleles of pho1were used, namely the previously described pho1-2 allele(Hamburger et al., 2002), having a nonsense point muta-tion in the fifth exon, and a new pho1-7 allele, having atransfer DNA in the second exon (Supplemental Fig. S1).

Quantitative PCR was performed on 4-week-old ro-settes of soil-grown Col-0 and pho1-7 and revealed that

Figure 1. (Continued.)bars) grown for 4 weeks in fertilized soil. For A to I, data are means6 SD of three samples obtained from independent plates, withthree technical replicates for each sample and each sample consisting of a pool of tissues isolated from 10 plants for shoots,approximately 50 plants for roots, and approximately 150 plants for root tips. For J, data are means 6 SD of three samples fromplants grown in independent pots and three technical replicates for each sample, with each sample being a pool of three plants.Asterisks denote statistical significance (*, P, 0.05; **, P, 0.01; and ***, P, 0.001) according to Student’s t test. Experimentswere performed independently three times with similar results.

Figure 2. Enhanced levels of JA and JA-Ile in Pi-deficient tissues. A, B, E, and F, JA (A and E) and JA-Ile (B and F) contents in leaves(A and B) and roots (E and F) from plants grown for 12 d in medium containing 1 mM or 100 mM Pi. C and D, JA (C) and JA-Ile (D)contents in shoots from plants grown for 12 d inmedium containing 1mM or 100mM Pi and 1 h after wounding. G to J, JA (G and I)and JA-Ile (H and J) in shoots of 4-week-old soil-grown Col-0, pho1-7, and pho1-2 plants sampled before wounding (G and H) or1 h after wounding (I and J). For A to F, data are means6 SD of five samples obtained from independent plates, with each sampleconsisting of a pool of tissues isolated from 20 plants for leaves and 100 plants for roots. For G to J, data are means 6 SD of fivesamples from plants grown in independent pots, with each sample being a pool of three plants. Asterisks denote statistical sig-nificance (*, P, 0.05; **, P, 0.01; and ***, P, 0.001) according to Student’s t test. For G to J, statistical analysis was performedcomparing Col-0 with each of the pho1mutants. Experiments were performed independently one time (I and J), two times (C, D,G, and H), or three times (A, B, E, and F) with similar results. FW, Fresh weight.



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the expression of JAZ10, VSP2, and LOX2was inducedin pho1-7 rosettes (Fig. 1J). Basal levels of JA and JA-Ilewere increased in leaves of both pho1-7 and pho1-2mutants compared with Col-0 (Fig. 2, G and H). Whilewounding of Col-0 rosettes resulted in expected in-creases in JA and JA-Ile levels, similar treatment of thepho1-7 and pho1-2 mutants resulted in 3- to 4-foldhyperaccumulation of both JA and JA-Ile relative toCol-0 (Fig. 2, I and J).

Induction of the JA Pathway by Pi Deficiency Is PartiallyControlled by the PHR1 Transcription Factor

The transcription factor PHR1 is known to play animportant role in the transcriptional activation of nu-merous genes by Pi deficiency and acts as a centralregulator of the Pi starvation response (Rubio et al.,2001; Bustos et al., 2010). The impact of PHR1 on theactivation of the JA pathway by Pi deficiency wasassessed by analyzing JAZ10 expression in the phr1mutant. While JAZ10 expression was enhanced 3-foldin seedlings of Col-0 grown for 7 d on Pi-deficient me-dium compared with Pi-sufficient medium, phr1-1 mu-tant seedlings showed no induction (Fig. 3A). However,after 10 d on Pi-deficientmedium, both Col-0 and phr1-1seedlings had a similar 7- to 8-fold induction of JAZ10. Incontrast, expression of the IPS1 gene, known to be di-rectly under the control of PHR1 (Rubio et al., 2001;Bustos et al., 2010), remained strongly down-regulatedin phr1-1 comparedwith Col-0 after both 7 and 10 d of Pideficiency (Fig. 3B). These results indicate that the ki-netics of the induction of the JA signaling pathway by Pideficiency is partially under the control of PHR1.

Contribution of the JA Pathway to Growth Phenotypes ofPi-Deficient Plants

Expression of the JA pathway in Arabidopsis isknown to lead to reductions in shoot growth (Zhangand Turner, 2008). Accordingly, under our growthconditions, rosettes of the aos and coi1-1mutant rosetteswere larger than those of Col-0 plants, with an increaseof approximately 25% in shoot fresh weight (Fig. 4, Aand B). To test whether JA accumulation was involvedin the reduction of pho1 shoot growth, double mutantsbetween pho1-7 and aos or coi1-1 were generated. ShootPi contents in the double mutants were similar to thoseof the pho1-7 parent and low compared with Col-0(Supplemental Fig. S2A). The shoot freshweight of bothpho1-7 aos and pho1-7 coi1-1 mutants was 2-fold highercompared with pho1-7 but remained 3-fold lower thanCol-0, while the petiole length of pho1-7 aos and pho1-7coi1-1was comparable to Col-0 and 3.5-fold longer thanthat for pho1-7 (Fig. 4, A and B; Supplemental Fig. S2B).Furthermore, while the pho1 mutant strongly accumu-lated anthocyanin in rosettes, a phenotype typicallyassociated with Pi deficiency, anthocyanin levels inthe double mutants pho1-7 aos and pho1-7 coi1-1 were

comparable to those of Col-0 plants (Fig. 4C). pho1-7 aosand pho1-7 coi1-1 double mutants also showed a sig-nificantly reduced induction of Pi starvation responsegenes, such as IPS1 and MGD3 (encoding a monoga-lactosyl diacylglycerol synthase involved in galactolipdsynthesis), in the rosettes compared with the pho1-7parent (Fig. 4, D and E). However, the increased pro-portions of sulfolipids and galactolipids relative tophospholipids, a biochemical phenotype typically as-sociated with Pi deficiency and observed in the shoot ofthe pho1mutant, were maintained in the pho1-7 aos andpho1-7 coi1-1 plants (Supplemental Fig. S3). JA and JA-Ile levels in the pho1-7 aos mutant were undetectable,and they were greatly reduced in the pho1-7 coi1-1 mu-tant compared with the pho1-7 parent (Fig. 4, F and G).Grafting experiments between roots and shoots ofpho1-7 and pho1-7 aos or pho1-7 coi1-1 showed that

Figure 3. The kinetics of JAZ10 induction by Pi deficiency is influencedby PHR1. JAZ10 (A) and IPS1 (B) expression is shown in Col-0 andphr1-1 seedlings grown for 7 or 10 d in medium containing 1 mM (graybars) or 20 mM (black bars) Pi. Data are means 6 SD of three samplesobtained from independent plates, with three technical replicates foreach sample and each sample consisting of a pool of tissues isolatedfrom 20 seedlings. Asterisks denote statistical significance (**, P, 0.01and ***, P, 0.001) according to Student’s t test. Statistical analysis wasdone comparing Col-0 and phr1-1 within each treatment. Experimentswere performed independently three times with similar results.


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Figure 4. Contribution of the JA pathway to the phenotype of Pi deficiency. A to E,Wild-type andmutant plants grown in fertilizedsoil for 4 weeks were compared for overall appearance (A), shoot fresh weight (B), shoot anthocyanin content (C), and the ex-pression of the IPS1 (D) and MGD3 (E) genes in shoots. In A, the pots containing Col-0 and pho1-7 plants at left are the same asthose shown at right. F andG, JA (F) and Ja-Ile (G) contents in rosette leaves from plants grown for 4 weeks in soil. H, The response



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perturbation of JA biosynthesis only in the shoot wassufficient to recapitulate the shoot phenotypes ofthe pho1-7 aos and pho1-7 coi1-1 double mutants(Supplemental Fig. S2C). Together, these results indi-cate that induction of the JA pathway in the shoot ofpho1 is partially responsible for several of the Pi star-vation phenotypes associated with this mutant.

While shoots of the pho1 mutant are Pi deficient, itsroots are Pi sufficient (Poirier et al., 1991). Since soil Pideficiency is first perceived by the root system, the ef-fect of JA accumulation was examined in plants expe-riencing Pi deficiency in both roots and shoots. Pideficiency leads to reduced growth of the primary root(Williamson et al., 2001; López-Bucio et al., 2002).Strong reduction of primary root elongation by Pi de-ficiencywas essentially maintained in the aos and coi1-34mutants, with only a small but statistically significantdifference observed compared with Col-0 (Fig. 4H). Atthe shoot level, Pi-deficient aos and coi1-34 showed 2- to3-fold reductions in anthocyanin accumulation relativeto Col-0 (Fig. 4I). Shoot fresh weight of the Arabidopsisaos and coi1-34 mutants was approximately 15% to 30%higher compared with Col-0 plants, irrespective ofwhether plantswere grown in high (1mM) or low (100 or20 mM) Pi (Fig. 4J).

Pi Deficiency Leads to Increased Resistance of Plants toInsect Herbivory

To investigate whether JA accumulation in pho1shoots had an effect on herbivory, we exposed plants tothe generalist insect larvae of S. littoralis. Only 30% ofthe caterpillars feeding on pho1-7 survived after 12 d,while the survival rate on Col-0, aos, and coi1-1, as wellas the double mutants pho1-7 aos and pho1-7 coi1-1, was90% to 100% (Fig. 5A). Surviving caterpillars feeding onpho1-7 showed a 3.5-fold reduction in fresh weightcompared with insects feeding on Col-0 (Fig. 5B). Asexpected, caterpillars feeding on the JA-deficient aosand coi1-1 mutants had higher fresh weight comparedwith Col-0 (Fig. 5B; Schlaeppi et al., 2008). The higher

resistance of pho1 to herbivory was greatly reduced inthe pho1-7 aos and pho1-7 coi1-1 double mutants (Fig.5B). However, caterpillars feeding on pho1-7 coi1-1 orpho1-7 aos double mutants showed a slightly reduced(1.4-fold) weight when compared with the parentalcontrols coi1-1 or aos. These data indicate that activationof the JA pathway is an important determinant affect-ing S. littoralis survival rate and larvae weight in thepho1 mutant but that JA-independent factors also in-fluence the weight of larvae feeding on pho1.

A bioassay was performed using the specialist her-bivore caterpillars of Pieris brassicae (Reymond et al.,2004). Glucosinolates are the characteristic herbivorydefensemetabolites of plants of the Brassicaceae family,to which Arabidopsis belongs, and these metabolitesare induced through the activity of the JA pathway(Mikkelsen et al., 2003). P. brassicae is equipped to de-toxify glucosinolates and, thus, shows similar feedingbehavior on Arabidopsis regardless of a plant’s JAstatus (Wittstock et al., 2004; Schlaeppi et al., 2008).P. brassicae caterpillars showed similar feeding behavioron pho1-7 and Col-0, with no significant difference ob-served in the fresh weight and survival rate of cater-pillars feeding on these plants (Fig. 5, C–E).

The effect of Pi deficiency on S. littoralis herbivorywas assessed in wild-type Arabidopsis as well as in twocrop plants, N. benthamiana and tomato. For all plants,both caterpillar weight and survival rate were reducedconsiderably in plants grown under Pi-deficient con-ditions compared with plants grown on Pi-sufficientconditions (Fig. 6). These results show that shoot Pideficiency leads to increased resistance to herbivory in adiversity of plants, including economically importantcrops.

DISCUSSION

Several phytohormones play important roles in theadaptation of plants to Pi deficiency, with most studiesfocusing on changes in the root system architecture(RSA; Niu et al., 2013). Under Pi deficiency, primary

Figure 4. (Continued.)of the primary root to low Pi was assessed by growing plants on agar-solidified medium containing 1 mM Pi for 7 d beforetransferring them to similar medium containing either 1 mM (gray bars) or 10mM (black bars) Pi for 3 d. Growth of the primary rootin the last 3 d was measured. I, Anthocyanin levels in shoots of plants grown for 7 d in agar-solidifiedmedium containing 1 mM Pifollowed by transfer to medium containing either 1 mM (gray bars) or 10 mM (black bars) Pi for 5 d. J, Fresh weight of the rosettes ofCol-0, aos, and coi1-34 plants grown for 18 d in agar-solidified medium containing 1 mM, 100 mM, or 20 mM Pi. For B, data aremeans6 SD of 20 plants grown in individual pots. For C, data aremeans6 SD of five plants grown in independent pots. For D and E,data are means6 SD of three samples from plants grown in independent pots and three technical replicates for each sample, witheach sample being a pool of three plants. For F andG, data aremeans6 SD of five samples from plants grown in independent pots,with each sample being an individual plant for Col-0, coi1-1, and aos and a pool of three plants for pho1-7, pho1-7 coi1-1, andpho1-7 aos. For H, data are means6 SD of 30 individual roots from three independent plates. For I and J, data are means6 SD of20 to 30 plants grown in five independent plates. For B to G, values marked with lowercase letters were statistically significantlydifferent from those for other groups marked with different letters (P, 0.05, ANOVAwith the Tukey-Kramer honestly significantdifference [HSD] test). n.d., Not detectable. For H to J, statistical analysis was done comparing Col-0 with the coi1-34 and aosmutants within each treatment, and asterisks denote statistical significance (*, P, 0.05 and **, P, 0.01) according to Student’st test. Experiments were performed independently one time (F, G, and J), two times (B, H, and I), or three times (C–E) with similarresults. FW, Fresh weight.


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root growth is inhibited, while the initiation andgrowth of both lateral roots and root hairs are stimu-lated. Changes in the synthesis, distribution, or sensi-tivity of the signaling pathway to various hormones areknown to act on the RSA (Rubio et al., 2009). For ex-ample, Pi deficiency is associated with increased sen-sitivity to auxin via the overexpression of the auxinreceptor TIR1 (Pérez-Torres et al., 2008), cytokininsynthesis and signaling via CRE1 are decreased(Franco-Zorrilla et al., 2002), and the synthesis of bothbrassinosteroids and gibberellins is decreased while thesynthesis of ethylene and strigolactones is increased(Ma et al., 2003; Jiang et al., 2007; Mayzlish-Gati et al.,2012; Singh et al., 2014). This work demonstrates thatPi deficiency leads to an increase in the synthesis ofJA/JA-Ile and to an activation of the JA signalingpathway in Arabidopsis. This was shown for bothroots and shoots of wild-type plants grown inmediumwith low Pi and in shoots of the pho1 mutant that ischronically Pi deficient due to a block in the transfer ofPi from roots to shoots.Although the absolute level in JA/JA-Ile present in

Pi-deficient pho1 leaves remained relatively low com-pared with wounded plants (approximately 40- to70-fold lower), pho1 had a robust hyperaccumulation of

JA/JA-Ile following wounding compared with the wildtype and an enhanced resistance to herbivory. This isanalogous to defense priming, which is defined as en-hanced activation of the defense response to insect orpathogen attack by a previous stimulation (Conrathet al., 2006; Frost et al., 2008). Priming of the herbivorydefense response has been associated with distinctsignaling cues, such as the release of volatiles fromneighboring wounded plants (Frost et al., 2008) or theassociation of roots with mycorrhizal fungi (Jung et al.,2012).

This work shows that Pi-deficient plants have in-creased resistance to S. littoralis herbivory. This wasshown for both the Pi-deficient pho1 mutant and Pi-deficient wild-type Arabidopsis, N. benthamiana, andtomato. The observation that the weights of larvaefeeding on pho1-7 coi1-1 and pho1-7 aos double mutantsare lower than those of larvae feeding on coi1-1 and aossingle mutants shows the involvement of JA-independentfactors affecting larvae weight on the pho1 mutant, in-dicating that the pathway(s) implicated in herbivoryresistance in pho1 and coi1/aos may not be the same.However, the larger difference in larvae weight be-tween pho1 and the wild type (3.5-fold) compared withcoi1-1 or aos and the respective pho1-7 coi1-1 and pho1-7

Figure 5. The pho1mutant is resistant to the generalist herbivore S. littoralis but not the specialist herbivore P. brassicae. A and B,Survival rate (A) and larvae weight (B) of S. littoralis caterpillars after 12 d of feeding on 4-week-old plants grown in fertilized soil.C and D, Survival rate (C) and larvae weight (D) of P. brassicae caterpillars after 7 d of feeding on 4-week-old plants grown infertilized soil. E, Representative images of Col-0 and pho1-7 plants after feeding on S. littoralis (top) and P. brassicae (bottom).Data aremeans6 SD of 20 to 30 caterpillars for A and B and 10 to 20 caterpillars for C andD feeding on individually grown plants.Values in A and B marked with lowercase letters were statistically significantly different from those for other groups marked withdifferent letters (P , 0.05, ANOVA with the Tukey-Kramer HSD test). For C and D, Student’s t test was used. Experiments wereperformed independently two times (C and D) or three times (A and B) with similar results.



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aos mutants (1.4-fold) suggests that the increased re-sistance of pho1 to herbivory is impacted by increasedJA levels. Considering that induction of the JA pathwayfollowing wounding provides a major contribution tothe resistance of plants to numerous herbivorous in-sects, including S. littoralis (Acosta and Farmer, 2010),the fact that wounded pho1 plants accumulate 3- to4-fold more JA-Ile and JA compared with woundedwild-type plants is likely to contribute to the impact ofthe pho1 mutation on herbivory resistance and larvaeweight.

Activation of the JA pathway by Pi deficiency hadeffects on some traits associated with Pi deficiency.Thus, in pho1-7 plants, blocking JA biosynthesis orsignaling via the aos or coi1-1 mutant, respectively,resulted in the elongation of petioles or the reduction ofleaf anthocyanin production. At the biochemical level,changes in lipid composition typically triggered by Pideficiency and observed in the pho1 mutant were notinfluenced by the JA pathway. Thus, the decrease inMGD3 expression observed in the pho1-7 coi1-1 andpho1-7 aos double mutants was not sufficient to impactmembrane lipid composition. Previous analysis of geneexpression in roots of the lpi4 mutant, which is insen-sitive to the inhibitory effect of Pi deficiency on primaryroot growth, revealed a down-regulation of severalgenes involved in JA biosynthesis, suggesting that JAmay contribute to RSA (Chacón-López et al., 2011). Ourdata show that the expression of JAZ10 and VSP2 isrobustly up-regulated in Pi-deficient root tips, indicat-ing that the increase in JA and JA-Ile levels observed inPi-deficient whole roots likely applies also to the roottip. Primary root elongation of the aos and coi1-34 mu-tants was still strongly reduced under Pi deficiency,with only a small but statistically significant differencecompared with wild-type plants, indicating a marginalcontribution of the JA pathway to this root phenotype.While blocking the JA pathway had a larger beneficialimpact on pho1 shoot growth than on the wild type, itdid not make the growth of pho1 comparable to that ofPi-sufficient wild-type plants. These results indicatethat, while JA levels in pho1 negatively affect shootgrowth, other factors associatedwith Pi deficiency havelarger contributions to this phenotype. In contrast topho1, blocking the JA pathway in plants experiencing Pideficiency in both roots and shoots had only marginaleffects on shoot growth. This may indicate that root-to-shoot long-distance signaling occurring in plants ex-periencing both root and shoot Pi deficiency may limitplant growth even in the absence of JA synthesis orperception. However, reduction of shoot anthocyaninstill occurred in aos and coi1-1 plants with low root andshoot Pi. This effect is likely mediated, in part, by theinteraction of the JAZ repressor with MYB75, a tran-scription factor activating the anthocyanin biosyntheticpathway (Qi et al., 2011), as well as through the inter-action between the JAZ and DELLA proteins (Jianget al., 2007; Hou et al., 2010).

The pathway linking Pi deficiency to JA inductionremains to be elucidated. While the kinetics of theactivation of the JA signaling pathway by Pi deficiencyin the phr1 mutant was delayed, activation of thepathway was not abolished, indicating that other sig-naling components in addition to PHR1 are partici-pating. Interestingly, Arabidopsis mutants in genesinvolved in inositol polyphosphate synthesis showalterations in the JA-mediated defense response toherbivorous insects (Mosblech et al., 2008, 2011; Lahaet al., 2015). The inositol polyphosphates InsP5 andInsP8 are known to bind to the COI1-JAZ coreceptorcomplex and likely act as coactivators (Sheard et al.,

Figure 6. Long-term Pi deficiency induces resistance to S. littoralisherbivory. Plants were grown from seeds on a soilless aeroponic systemsupplemented with Hoagland nutrient solution containing either 1 mMPi or no added Pi. Weight (left) and survival rate (right) of S. littoraliscaterpillars were measured after either 12 d of feeding on 4-week-oldArabidopsis (A) or 12 d of feeding on 3-week-old tomato (B) or N.benthamiana (C). Data are means 6 SD of 10 to 20 caterpillars feedingon individually grown plants. Student’s t test was performed, and as-terisks denote statistical significance (*, P, 0.05; **, P, 0.01; and ***,P , 0.001). Experiments were performed independently two times(B and C) or three times (A) with similar results.


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2010; Laha et al., 2015). The ipk1-1 mutant deficient inInsP6 synthesis also is affected in the Pi signaling cas-cade (Stevensons-Paulik et al., 2005; Kuo et al., 2014). Itis thus possible that Pi deficiency influences both Pi andJA signaling pathways via the modulation of the inositolpolyphosphate pool. Unfortunately, no studies have spe-cifically measured changes in the inositol polyphosphatepools in response to Pi deficiency in plants.A few other examples of interactions between nutri-

ents and the JA pathway have been described. Theaddition of silicon was shown to trigger priming of theJA-mediated herbivory defense in rice (Ye et al., 2013).Potassium deficiency in Arabidopsis increased the ex-pression of several JA-responsive genes, enhanced thesynthesis of several oxylipins in leaves, including JA,and was associated with reduced damage by thrips(Armengaud et al., 2004, 2010; Troufflard et al., 2010).Sulfur deficiency resulted in an enhanced expression ofseveral genes involved in JA synthesis, and mutationaffecting sulfur metabolism influenced basal JA levels(Hirai et al., 2003; Maruyama-Nakashita et al., 2003;Nikiforova et al., 2003; Rodríguez et al., 2010).Insect herbivory and Pi deficiency are two stresses

that individually have negative impacts on plant fitnessand seed yield and that are prevalent in both naturalplant ecosystems and agriculture. Furthermore, insectherbivory has been shown to impact ecological andevolutionary changes in plant populations, even over afew generations (Maron and Crone, 2006; Agrawalet al., 2012). As the JA-mediated wound response isimplicated in the resistance to herbivory not only byinsects but also by detritivorous crustaceans and ver-tebrate herbivores (Farmer and Dubugnon, 2009; Mafliet al., 2012), the activation of the JA pathway by Pideficiency may have broad ecological and evolutionaryconsequence on plant populations. Our work providesan insight into how nutrient supply can impact thegrowth and defense of plants, an issue of global im-portance, since Pi-based fertilizers and plant pestprotection are the two major inputs of modern-dayagriculture.

MATERIALS AND METHODS

Plant Material and Growth Conditions

All Arabidopsis (Arabidopsis thaliana) plants used in this study, includingmutants and transgenic plants, were in the Col-0 ecotype. The aos, coi1-1, coi1-34,and pho1-2 mutants were described previously (Feys et al., 1994; Hamburgeret al., 2002; Park et al., 2002; Acosta et al., 2013). The gl1mutation present in theoriginal coi1-1mutant (Feys et al., 1994) was removed by backcrossing and wasused in all experiments (provided by Jane Glazebrook, University of Minne-sota). The pho1-7 mutant is a transfer DNA mutant from the Salk collectionand was obtained from the European Arabidopsis Stock Centre (http://arabidopsis.info/). For in vitro experiments, plants were grown in one-half-strength Murashige and Skoog (MS) salts (Duchefa) containing 1% (w/v) Sucand 0.7% (w/v) agar. For Pi-deficient medium, MS salts without Pi (Caisson)and purified agar (Conda) were used. Phosphate buffer at pH 5.7 (93.5%KH2PO4 and 6.5% K2HPO4) was used to adjust Pi concentration in medium. Inthe Pi-deficient medium, phosphate buffer was replaced by equimolar amountsof KCl. Growth chamber conditions were 22°C, 60% humidity, 100 mE m22 s21

white light, and a photoperiod of either 16 h of light/8 h of dark (long days) or10 h of light/14 h of dark (short days).

An original pho1-7 coi1-1 double homozygous mutant was identifiedthrough genotyping for the pho1-7 and coi1-1 alleles and the correspondingwild-type alleles (a list of primers used in this work is found in SupplementalTable S1). Genotyping for coi1-1 includes sequencing of the PCR fragment toidentify the point mutation. Since the pho1-7 coi1-1 double homozygous mutantismale sterile, it was crossedwith pollen from fertile pho1-7/pho1-7 coi1-1/COI1plants, resulting in the generation of a seed stock that is 50% pho1-7/pho1-7coi1-1/COI1 and 50% pho1-7/pho1-7 coi1-1/coi1-1. In all experiments involv-ing the pho1-7 coi1-1 double homozygous mutant, plants from this mixed seedstock were first grown and assayed for various parameters (herbivory test,growth parameters, anthocyanin quantification, etc.) and were subsequentlygenotyped after sampling to identify plants that were pho1-7 coi1-1 doublehomozygous.

Quantitative Reverse Transcription-PCR

Plantmaterialwas immediately frozen in liquid nitrogen and groundusing amortar and pestle. Approximately 100 to 150 mg of powder was used to extractthe RNA using the RNeasy plant mini kit (Qiagen). Samples were treated withDNase on columns during RNA extraction using the RNase-free DNase kit(Qiagen). Approximately 500 ng of RNA per sample was used for reversetranscription using SuperScript II reverse transcriptase (Invitrogen). Quantita-tive PCR was performed using SYBR select master mix (Invitrogen) and Stra-tagene thermocyclerMx3005p. Primers used for quantitative PCR are describedin Supplemental Table S1.

Anthocyanin and Lipid Quantification

Anthocyanin quantification was essentially performed as described pre-viously (Mita et al., 1997). In brief, 25 mL ofmethanol containing 1% (v/v) HClwas added per 1 mg of leaf fresh weight. Leaves were then homogenized inthe solution overnight at 4°C. Samples were centrifuged, and the absorbanceof the supernatant was measured at 657 and 530 nm. Anthocyanin was cal-culated according to the formula A530 2 (0.25 3 A657), to compensate for thecontribution of chlorophyll and its degradation products. Analysis of diac-ylglycerides was performed by the Kansas Lipidomics Research Center(www.k-state.edu/lipid/lipidomics/).

Insect Bioassays

Bioassays with Spodoptera littoralis and Pieris brassicae were performed asdescribed previously (Bodenhausen and Reymond, 2007). In brief, plants wereplaced in cages fitted with windows on both sides or in nylon tents (Bugdorm;http://shop.bugdorm.com), and freshly hatched insects were placed directlyon leaves. S. littoralis and P. brassicae caterpillars were left to feed for 12 and 7 d,respectively. Insects were weighed on the Mettler-Toledo MT5 precision bal-ance. S. littoralis eggs were obtained from Syngenta, while P. brassicae werereared in house as described previously (Schlaeppi et al., 2008).

Pi Measurement

Pi measurement was performed as described previously (Rouached et al.,2011). In brief, 50 mL of water was added per 1 mg fresh weight of material.Material was frozen at 220°C and then thawed at ambient temperature beforeboiling at 80°C for 1 h. Supernatant was taken for Pi measurement after cen-trifugation. Pi measurement was done using the molybdate assay as describedpreviously (Ames, 1966).

Hormone Quantification

JA and JA-Ile quantification was performed as described previously(Glauser et al., 2014). In brief, plant material was frozen in liquid nitrogen im-mediately after harvesting. Samples were then ground using a mortar andpestle, and approximately 100mg of the powder was weighed using a precisionbalance (Mettler Toledo AG245). JA and JA-Ile were extracted in a mixture ofethylacetate:formic acid (99.5:0.5, v/v) containing d5-JA (1 ng) and [13C6]JA-Ile(1 ng) as internal standards. The solution was then evaporated to dryness, andthe residuewas reconstituted inmethanol:water (70:30, v/v). Fivemicroliters ofextract was injected into an ultra-high-pressure liquid chromatography system



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coupled to a tandem mass spectrometer. Quantification was achieved using afive-point calibration curve ranging from 0.5 to 200 ng mL21 JA and JA-Ile andcontaining 1 ng of labeled internal standards.

Root Assay

Seedlings were grown vertically in one-half-strength MS salts containing1% (w/v) Suc and 0.7% (w/v) agar for 7 d. Seedlings were then transferred toplates containing one-half-strength MS salts with either 1 mM Pi medium (highPi) or 10 mM Pi medium (low Pi). Root length after the transfer was measured3 d later using ImageJ software.

Statistical Analysis

Data were generated from material grown in different Petri dishes and in-dividual pots at different times and are reported as means with SD. Statisticalsignificance was evaluated using either Student’s t test or ANOVA with theTukey-Kramer HSD test. Experiments also were independently reproducedwith a gap of several months, with consistent results, as indicated in the figurelegends.

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. The pho1 alleles.

Supplemental Figure S2. Contribution of the JA pathway to the phenotypeof Pi deficiency.

Supplemental Figure S3. Lipid profile of leaves of plants grown in soil.

Supplemental Table S1. List of oligonucleotides.

ACKNOWLEDGMENTS

We thank Philippe Reymond and Edward Farmer for useful discussionsand critical reading of the article, Christelle Bonnet and Steve Lassueur forhelp on caterpillar bioassays, and Javier Paz-Ares for providing seeds of thephr1 mutant.

Received February 19, 2016; accepted March 24, 2016; published March 25,2016.

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Phosphate Deﬁciency Induces the Jasmonate Pathway and...genes (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007).Uponwounding,enhancedJA-Ilelevelspromote the degradation