auxin and strigolactone signaling are required for ... · moving down the primary shoot would be...

Auxin and Strigolactone Signaling Are Required forModulation of Arabidopsis Shoot Branching byNitrogen Supply1[W][OPEN]

Maaike de Jong2, Gilu George2, Veronica Ongaro2, Lisa Williamson, Barbara Willetts, Karin Ljung,Hayley McCulloch, and Ottoline Leyser*

Sainsbury Laboratory, University of Cambridge, Cambridge CB2 1LR, United Kingdom (M.d.J., H.M., O.L.);Department of Biology, University of York, York YO10 5DD, United Kingdom (G.G., V.O., L.W., B.W., O.L.);and Umeå Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University ofAgricultural Sciences, SE–901 83 Umea, Sweden (K.L.)

The degree of shoot branching is strongly affected by environmental conditions, such as nutrient availability. Here wedemonstrate that nitrate limitation reduces shoot branching in Arabidopsis (Arabidopsis thaliana) both by delaying axillary budactivation and by attenuating the basipetal sequence of bud activation that is triggered following floral transition. Ammoniumsupply has similar effects, suggesting that they are caused by plant nitrogen (N) status, rather than direct nitrate signaling. Weidentify increased auxin export from active shoot apices, resulting in increased auxin in the polar auxin transport stream of themain stem, as a likely cause for the suppression of basal branches. Consistent with this idea, in the auxin response mutant axr1and the strigolactone biosynthesis mutant more axillary growth1, increased retention of basal branches on low N is associated witha failure to increase auxin in the main stem. The complex interactions between the hormones that regulate branching make itdifficult to rule out other mechanisms of N action, such as up-regulation of strigolactone synthesis. However, the proposedincrease in auxin export from active buds can also explain how reduced shoot branching is achieved without compromising rootgrowth, leading to the characteristic shift in relative biomass allocation to the root when N is limiting.

Plants continuously adjust their development to suitthe environmental conditions in which they are grow-ing. This spectacular developmental plasticity meansthat plants with identical genotypes can have verydifferent morphologies. A good example of such de-velopmental plasticity is the degree of shoot branching(Leyser, 2009). Shoot branches develop from axillarymeristems laid down in the axils of the leaves pro-duced by the primary shoot apical meristem. Theseaxillary meristems can arrest after producing a fewleaves to form a small dormant bud, or they can re-main active or later reactivate to produce a branch. Theleaves produced on the branch also harbor axillary

meristems, allowing higher order branching. Hence, de-pending on the activity of axillary meristems, the adultplant can range in form from a single unbranched stemto a highly ramified bush. Many environmental factorsare integrated to regulate the dormancy-activity transi-tions of axillary meristems. Prominent among these is thesupply of mineral nutrients such as inorganic phosphateand nitrate (NO3

2; Domagalska and Leyser, 2011; Breweret al., 2013).

Limited NO32 availability can have profound effects

on both root and shoot system architecture and bio-mass allocation, shifting the balance in favor of theroot. In the root, low NO3

2 can promote the elongationof both primary and lateral roots relative to shoot dryweight (Scheible et al., 1997; Zhang et al., 1999; Linkohret al., 2002). Furthermore, a patch of high NO3

2 canstimulate local proliferation of lateral roots into thepatch and can suppress root growth outside the patch(Drew, 1975; Scheible et al., 1997; Zhang and Forde,1998; Zhang et al., 1999; Linkohr et al., 2002). Some ofthese responses, such as the proliferation of roots into ahigh-NO3

2 patch, are the result of direct local NO32

signaling, but there is also clear evidence for systemicnitrogen (N) status effects. In particular, both the in-hibitory effect on root growth by high NO3

2 supplyand the stimulatory effect of low NO3

2 appear to bemediated by shoot N status (Scheible et al., 1997;Zhang et al., 1999). Thus, variation in NO3

2 avail-ability triggers systemically coordinated changes inroot and shoot system architecture.

1 This work was supported by the European Research Council(grant no. 294514 EnCoDe to O.L., M.d.J., and H.M.), the GatsbyFoundation (to O.L.), the Swedish Governmental Agency for Innova-tion Systems and the Swedish Research Council (to K.L.), a BurgessStudentship (to G.G.), the Biotechnology and Biological Sciences Re-search Council (to O.L., V.O., and B.W.), and Norsk Hydro (to O.L.and L.W.).

2 These authors contributed equally to the article.* 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:Ottoline Leyser ([email protected]).

[W] The online version of this article contains Web-only data.[OPEN] Articles can be viewed online without a subscription.www.plantphysiol.org/cgi/doi/10.1104/pp.114.242388

384 Plant Physiology�, September 2014, Vol. 166, pp. 384–395, www.plantphysiol.org � 2014 American Society of Plant Biologists. All Rights Reserved.

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There has been considerable progress in understand-ing how local NO3

2-regulated changes in the root sys-tem are effected (for review, see Bouguyon et al., 2012),but much less is known about how NO3

2 availabilityregulates shoot branching and how this is coordinatedwith the root. These are important questions becauseof the agricultural imperative to reduce fertilizer in-puts while maintaining yield. With respect to shootbranching, there is little physiological evidence for adirect role for mineral nutrients in bud activation, be-cause although the sustained growth of branches un-doubtedly requires a nutrient supply, application ofnutrients to dormant buds does not usually activatetheir growth (Cline, 1991). In contrast, there is ampleevidence that hormones regulate bud activity, and thesesame hormones are excellent candidates to act as sys-temic coordinators of nutrient signaling (Leyser, 2009).This suggests the hypothesis that NO3

2-triggered changesin shoot system architecture are mediated by changesin hormone activity.There is good evidence that NO3

2 availability in theroot system positively regulates cytokinin (CK) bio-synthesis (Takei et al., 2002, 2004; Miyawaki et al.,2004; Wang et al., 2004) and that this is required for atleast some systemic responses to NO3

2 availability(Ruffel et al., 2011). Two additional systemically mo-bile hormones have been strongly implicated in shootbranching control, namely auxin and strigolactones (SLs),both of which are generally considered to inhibit budgrowth (for review, see Dun et al., 2009; Domagalska andLeyser, 2011).Auxin is mainly produced in the young expanding

leaves at the shoot apex (Ljung et al., 2001). It istransported down the stem in the polar auxin trans-port stream (PATS) and inhibits bud growth. In Ara-bidopsis (Arabidopsis thaliana), this directional flowinvolves the PIN-FORMED1 (PIN1) protein, a memberof a family of auxin efflux proteins that is localized tothe basal membrane of cells of the PATS (Okada et al.,1991; Gälweiler et al., 1998; Wisniewska et al., 2006).Auxin moving in the PATS does not enter the bud inappreciable amounts, indicating an indirect mode ofaction (Brown et al., 1979; Everat-Bourbouloux andBonnemain, 1980; Prasad et al., 1993; Booker et al.,2003). There is good evidence to suggest that auxintransport in the main stem inhibits bud outgrowth bypreventing axillary buds from establishing their ownPATS out into the main stem (Li and Bangerth, 1999;Bennett et al., 2006; Prusinkiewicz et al., 2009; Ballaet al., 2011; Shinohara et al., 2013). In addition, auxinmay act by regulating the production of one or moresecond messengers in the stem, which move up intothe bud to regulate bud activity locally and directly(Snow, 1937). Both CK and SL are good candidatesecond messengers, since the transcription of the bio-synthetic genes for both these hormones is regulatedby auxin (Nordström et al., 2004; Foo et al., 2005;Johnson et al., 2006; Hayward et al., 2009).SLs are synthesized in both the root and shoot and

are transported acropetally in the transpiration stream

(Beveridge et al., 1996; Napoli, 1996; Booker et al.,2004; Kohlen et al., 2011). SLs may directly and locallyinhibit bud activation, as mentioned above (Breweret al., 2009; Dun et al., 2012, 2013), and/or they mayact systemically to influence bud growth by modulatingPIN1 accumulation at the plasma membrane and, hence,the establishment of canalization of auxin transportout of the bud (Bennett et al., 2006; Prusinkiewiczet al., 2009; Crawford et al., 2010; Shinohara et al.,2013).

There is limited evidence about the roles of SLs andauxin in the control of shoot branching in response toN. SL exudation from the root can be up-regulated byN deficiency, and it has been demonstrated that SLsare required for the suppression of branching underphosphate deficiency, which also elevates SL biosyn-thesis and exudation from the root (Yoneyama et al.,2007a, 2012; López-Ráez et al., 2008; Umehara et al.,2010; Kohlen et al., 2011; Mayzlish-Gati et al., 2012;Foo et al., 2013). In the case of auxin, increased auxinmoving down the primary shoot would be predictedto suppress shoot branching, and there is some evi-dence to support such an increase from studies of rootresponses to N. For example, in maize (Zea mays),shoot-derived auxin appears to enhance root branch-ing responses to N deprivation (Tian et al., 2008; Liuet al., 2010). Similarly, changes in shoot-to-root auxintransport have been implicated in the suppression ofnodulation in response to N sufficiency; however,there are apparently contradictory results as to whethernodule suppression results from high or low shoot-to-root auxin transport (van Noorden et al., 2006; Jin et al.,2012).

Here, we present work aimed at testing the hy-pothesis that auxin and SLs are important mediators inshoot branching suppression under low-NO3

2 condi-tions, making use of auxin and SL mutants of Arabi-dopsis.

RESULTS

Low NO32 Restricts Shoot Branching in Arabidopsis by

Early Termination of the Basipetal Sequence ofBud Activation

In Arabidopsis, the primary shoot apical meristemremains indeterminate, producing leaves at its flankswith usually a single axillary meristem (AM) in eachaxil. During the vegetative phase, the AMs are slow todevelop and cannot be morphologically identified formany plastochrons below the primary shoot apicalmeristem. This means that in long-day conditions, thereare usually no active branches produced in the vege-tative phase. After the floral transition, AMs arise rap-idly in the axils of all leaves. These AMs activate toform lateral inflorescences in a basipetal sequence thatusually includes all the inflorescence-borne caulineleaves and proceeds down into the rosette (Hempeland Feldman, 1994; Stirnberg et al., 1999; Grbi�c andBleecker, 2000).

Plant Physiol. Vol. 166, 2014 385

Modulation of Shoot Branching by Nitrogen Supply



To investigate the effect of N supply on branching,we grew Arabidopsis plants with NO3

2 supply rangingfrom 9 to 1.8 mM. Across this range, the mean totalnumber of secondary shoots produced dropped from4.25 on 9 mM NO3

2 to 2.9 on 1.8 mM NO32 (Fig. 1A).

Measurement of free NO32 in the leaves of 4-week-old

plants revealed clear evidence of reduced NO32 levels in

the leaves of plants supplied with 2.25 mM NO32 or less.

Importantly, even plants grown on 1.8 mM NO32 still

showed substantial accumulation of free NO32 during

the vegetative phase, suggesting N limitation ratherthan N starvation (Fig. 1B). Based on these results, 9 and1.8 mM NO3

2 supply were selected as the standardN-sufficient and N-limited conditions for further analysis.

To characterize in more detail the effect of low NO32

on branching, the lengths of the three most apicalbranches were followed over time after anthesis ofthe first flower on the primary inflorescence. With9 mM NO3

2 supply, these three branches activatednearly simultaneously and elongated rapidly, reaching

lengths in excess of 12 cm over the time course of theexperiment (Fig. 2A). On 1.8 mM NO3

2, bud activationand elongation were delayed, and the basipetal se-quence of activation was much more obvious. By6 DPA, only the most apical bud had elongated sig-nificantly. By 11 d, the second bud was also activelygrowing, whereas there was still no significant elon-gation of the third bud (Fig. 2B). Even the first budelongated more slowly than its counterpart on 9 mM

NO32, reaching only approximately 4 cm by the end

of the experiment. Thus, reduced NO32 availability re-

sulted in delayed bud activation, reduced bud elonga-tion, and early termination of the basipetal activationsequence, leading to reduced total branch number(Figs. 1A, and 2).

Branch Suppression Is Not a Direct Effect of NO32

Signaling

There is good evidence that the NO32 ion itself can

act as a signal (Crawford, 1995; Stitt, 1999; Forde, 2002;Takei et al., 2002). To investigate whether NO3

2 signalsdirectly to promote branching, plants were grown onlimiting NO3

2 supplemented with increasing amountsof ammonium (NH4

+) as an N source. This approachwas adopted because using NH4

+ as the sole N sourcewas found to be toxic to Arabidopsis. Plants suppliedwith only 1 mM NO3

2 produced a mean of approxi-mately one secondary shoot, while the number ofbranches was double for plants supplied with both1 mM NO3

2 and 1 mM NH4+. This mean increased

slightly, but not significantly, under 2 mM NH4+ with

1 mM NO32 and 3 mM NO3

2 supply (Fig. 3A). Similarresults were obtained when plants were grown understerile conditions (Supplemental Fig. S1), demonstrat-ing that the effects are unlikely to be due to microbialconversion of NH4

+ to NO32.

These findings suggest that branching is not directlytriggered by NO3

2 acting as a signal but that the re-sponse relates to the overall N status of the plant. Toinvestigate this idea further, the root-total mass ratio ofthese plants was examined, since low nutrient avail-ability is well known to trigger the redistribution ofrelative growth from shoots to roots. As expected, re-duced branching is accompanied by increased pro-portional biomass allocation to roots, regardless ofwhether N was supplied as NO3

2 alone or as an NH4+ +

NO32 mix (Fig. 3B).

Auxin and SL Are Both Involved in the BranchingResponse to N

Since NO32 is apparently not directly involved

in the shoot branching response to N nutrition, wetested whether auxin or SL is required. The responseof branching to N limitation in wild-type plants wascompared with those of the auxin-resistant mutantaxr1-3 (Lincoln et al., 1990) and the SL biosynthesis

Figure 1. Effects of NO32 supply on shoot branching and leaf NO3

2

concentration. A, Mean number of secondary shoots of wild-typeplants grown on NO3

2 concentrations ranging from 9 to 1.8 mM. Thenumber of branches of 5 mm or more was determined when the pri-mary apex had ceased activity. Data are means 6 SE of eight plants.Different letters indicate statistically significant differences (P , 0.05,by Kruskal-Wallis H test). B, Leaf NO3

2 concentrations of wild-typeplants grown on NO3

2 concentrations ranging from 9 to 1.8 mM. NO32

measurements were conducted on leaves collected from 4-week-oldplants just prior to bolting. Data are means 6 SE of eight plants. Dif-ferent letters indicate statistically significant differences (P , 0.05, byANOVA). FW, Fresh weight.

386 Plant Physiol. Vol. 166, 2014

de Jong et al.


http://www.plantphysiol.org/cgi/content/full/pp.114.242388/DC1


mutant more axillary growth1-1 (max1-1) (Stirnberg et al.,2002; Booker et al., 2005). Both mutants responded toN limitation by reducing their branching, although bothmutants still produced more secondary shoots thanwild-type plants under the same N-limiting conditions(Fig. 4A). Similar results were obtained for the SL bio-synthesis mutants max3 and max4 and for the SL sig-naling mutant max2 (Supplemental Fig. S2). In the axr1-3max1-1 double mutant, there was no significant dif-ference in branch numbers on high and low NO3

2 inthis experiment (Fig. 4A). There was some variabilitybetween experiments in the extent of the response in thedouble mutant (Supplemental Fig. S3); nonetheless,these results suggest that the ability to reduce branchingin response to low NO3

2 is at least partially dependenton auxin and SL signaling.It was apparent from these experiments that N dep-

rivation also results in reduced stature (Fig. 4, B and C).Interestingly, this effect was abolished in the axr1 mu-tants but was unaffected by mutation in max1, sug-gesting that it is auxin dependent but SL independent.

N Deprivation and SL Treatment Have Similar Effects onBud Growth

The reduced ability of the max mutants to suppressbranching on low N suggests that either their buds are

nearly constitutively active or that bud suppression onlow N is partly mediated by dynamic changes in SLlevels. Direct assay of SLs, especially in Arabidopsisshoots, is currently extremely challenging. Therefore,we assessed whether low NO3

2 has SL-like effectson the shoot, using a well-established two-bud assay(Ongaro et al., 2008). In this assay, stem segments car-rying two nodes are excised from plants and sup-plied with nutrient solution basally, which can beadjusted to include different hormones or nutrients, asrequired. The growth of the two buds is monitoredover time, and the relative growth index (RGI) of thetwo buds can be determined. RGI is the length of thelongest bud divided by the total length of both buds.Usually, either one bud grows vigorously and theother does not, giving an RGI close to 1, or both budsgrow equally well, giving an RGI close to 0.5. The

Figure 3. Comparison of the effects of NH4+ and NO3

2 as N sourceson shoot branching and root fraction. Mean numbers of secondaryshoots (A) and root fraction (B) of wild-type plants grown with differentN supplies are shown. Plants were grown in 10% soil and 90% sandsupplemented with nutrient solutions containing 1 mM NO3

2, 1 mM

NH4+ + 1 mM NO3

2, 2 mM NH4+ + 1 mM NO3

2, or 3 mM NO32. Plants

were grown in 3-inch pots with four plants per pot and eight pots pertreatment. Data were collected at 6 weeks, when the first seeds wereripe. For shoot number (A), branches of 5 mm or more were counted.Data are means 6 SE of 32 plants. Different letters indicate statisticallysignificant differences (P , 0.05, by Kruskal-Wallis H test). The rootfraction (B) was calculated per pot as the dry weight of the roots di-vided by the total dry weight of the four plants. Data are means6 SE ofeight replicates. Different letters indicate statistically significant dif-ferences (P , 0.05, by ANOVA).

Figure 2. Effects of NO32 supply on the timing of bud activation.

A, Mean branch length from the three most apical nodes (N1, N2, andN3) of wild-type plants grown on 9 mM NO3

2 at anthesis and 2, 6, and11 DPA. N1 is the uppermost node. B, Mean branch length from thethree most apical nodes of wild-type plants grown on 1.8 mM NO3

2 atanthesis and 2, 6, and 11 DPA. Data are means 6 SE of six to 18 plants.







mean RGI thus captures the relative frequency of theseoutcomes. In this assay, SLs have been shown to shiftthe RGI toward 1 (Crawford et al., 2010). To assess theeffect of NO3

2 availability on RGI, we grew wild-typeand SL-resistant mutant (max2) plants on 9 or 1.8 mM

NO32, excised a two-node segment from their bolting

stems, and measured RGIs under high and low NO32,

respectively, with or without basal addition of thesynthetic SL, GR24. As reported previously, the RGI ofwild-type explants under N-sufficient conditions was

significantly higher than that of max2 mutants, andGR24 treatment increased the wild-type RGI furtherbut had no effect on that of max2 (Fig. 5A). The effect oflow N supply resembled GR24 treatment. Low N sig-nificantly increased the RGI of wild-type explants buthad no significant effect on the RGI of max2 mutants(Fig. 5A). GR24 had no additional effect when suppliedto low-NO3

2-grown plants; however, the wild-typeRGIs under both these treatments were nearly maxi-mal. In these experiments, the wild-type RGI inN-sufficient conditions was, on average, 0.8, whichis higher than that observed typically (Ongaro et al.,2008; Crawford et al., 2010). This may be because toallow full control of N supply the plants were grownon a sand and Terragreen mix, rather than on compost.

This result is consistent with the hypothesis thatshoot branching suppression on low NO3

2 is partlyattributable to increased SL production, as suggestedby the observation that low NO3

2 increases SL levelsin root exudates from some species (Yoneyama et al.,2007a, 2012, 2013; Jamil et al., 2011; Foo et al., 2013).However, it is equally consistent with other hypothe-ses, such as increased auxin production on low NO3

2.To assess further whether low NO3

2 increases SLlevels, and in particular whether this is due to increasedtranscript abundance for SL biosynthetic genes, weexamined publicly available expression data for thesegenes (ArrayExpress; Rustici et al., 2013). To date,four such genes are known: MAX1, MAX3, MAX4,and DWARF27. Among six experiments examiningthe effects of NO3

2 supply or resupply after NO32

deprivation (Wang et al., 2003; Scheible et al., 2004;Gifford et al., 2008, 2013; Patterson et al., 2010; Krappet al., 2011), we found no evidence of a consistenteffect of N supply on the transcription of any of thesegenes (Supplemental Table S1). The transcript abun-dance of core genes in the SL signaling pathway,DWARF14 and MAX2, was also not affected (Sup-plemental Table S1).

Auxin Transport in the Main Stem Is Unaffected byN Availability

To probe further the possibility of changes in SLlevels on low N, we assessed auxin transport changes.A signature of GR24 treatment and increased endog-enous SL synthesis is reduced polar auxin transport inthe main stem (Crawford et al., 2010; Shinohara et al.,2013). This effect is associated with a rapid SL-induceddepletion in PIN1 accumulation on the plasma mem-brane of xylem parenchyma cells in the stem. Depletionof PIN1 by SL can occur within 10 min, independentlyof new transcription, and therefore is a primary SLresponse (Shinohara et al., 2013). To assess the ef-fects of low NO3

2 on auxin transport through thestem, radiolabeled auxin was applied to the apical endof stem segments excised from plants grown with lowor high NO3

2 supply, and the amount transportedto the basal end was measured. As demonstrated

Figure 4. Responses of auxin signaling and SL biosynthesis mutants toNO3

2 supply. A and B, Mean number of secondary shoots (A) andprimary shoot length (B) of 6-week-old wild-type plants (WT), auxin-resistant axr1-3 mutants, SL biosynthesis max1-1 mutants, and axr1-3max1-1 double mutants grown on either 9 or 1.8 mM NO3

2. Data aremeans6 SE of 15 to 16 plants. Asterisks indicate statistically significantdifferences between the two NO3

2 treatments (P , 0.05) as assessedusing a Mann-Whitney U test (A) and a Student’s t test (B). C, Images ofrepresentative plants of each genotype grown on 9 mM (high [H]) or1.8 mM (low [L]) NO3

2.


de Jong et al.






previously, reduced SL levels in the max1 mutantresulted in increased auxin transport, but mutation inaxr1 had no effect (Bennett et al., 2006; Prusinkiewiczet al., 2009; Crawford et al., 2010). NO3

2 supply hadno significant effect on auxin transport in any of thegenotypes assessed (Fig. 5B), and accumulation ofPIN1 on the plasma membrane of xylem parenchymacells in the stem was also unaffected by NO3

2 supply(Fig. 5C). These results argue against the hypothesisthat reduced NO3

2 supply increases endogenous SLlevels, consistent with the lack of effect on transcript

abundance of SL biosynthetic genes. More generally,the results suggest that changes in auxin transport arenot involved in the branching response to NO3

2

supply.

Reduced N Availability Increases Auxin Levels in theMain Stem PATS

As mentioned above, an alternative hypothesis forbranch inhibition by low NO3

2 is that N limitationincreases auxin levels in the polar transport stream inthe shoot. To test this hypothesis, we measured theamount of auxin moving in the PATS of stem segmentsof the various genotypes under investigation by col-lecting the auxin that emerged from the base of thestem segments over a 24-h period and subjecting theexudate to mass spectrometric analysis. Auxin exu-dates from stem segments taken from either the apicalpart of the bolting stem, between the most apicalcauline node and the first silique, or the basal part ofthe bolting stem, between the most basal cauline nodeand the rosette, were assayed. In wild-type plantsgrown on low NO3

2, the amount of auxin exportedfrom apical stem segments was double that of plantsgrown on high NO3

2 (Fig. 6A, inset). For basal stemsegments from plants grown on high NO3

2, theamount of auxin exported was greater than that ex-ported from apical segments, as reported previously(Prusinkiewicz et al., 2009). This is likely due to theincreased number of active shoots feeding auxin intothe basal part of the stem (Prusinkiewicz et al., 2009).However, there was only a small nonsignificant in-crease in auxin in the basal stem segments of low-NO3

2-grown plants compared with those grown onhigh NO3

2 (Fig. 6A, inset), consistent with our obser-vation that the buds of the more basal nodes on thebolting stem on low NO3

2 remain dormant and thusdo not export auxin (Figs. 2B and 4C).

As reported previously, both axr1 mutants and max1mutants have more auxin in the PATS than the wildtype, although in this experiment not all these differ-ences are statistically significant (Fig. 6; Bennett et al.,2006; Prusinkiewicz et al., 2009). The amount of auxinexported from apical stem segments of the doublemutant exceeds that from both single mutants. In basalstem segments, max1 exports the most auxin, with asubstantial increase compared with the apical segments.The mutant backgrounds involving axr1 show littleincrease in auxin levels over those exported from ap-ical segments, as reported previously (Prusinkiewiczet al., 2009).

In contrast to the wild type, the amount of auxinexported from the apical stem segments of the variousmutants was not significantly affected by NO3

2 supply(Fig. 6). These results support the idea that reducedbranching on low NO3

2 is caused at least in part byincreased auxin export from each active apex. Thislow-NO3

2-induced increase in auxin is dependent onauxin signaling and SLs.

Figure 5. Effects of NO32 supply on SL-related phenotypes. A, RGI of

the buds from two-node explants of wild-type plants (WT) and themax2 SL signaling mutant grown on 9 or 1.8 mM NO3

2, with orwithout 1 mM GR24 treatment, as indicated. The data represent the RGI7 d after excision, which is calculated as the length of the longestbranch divided by the total length of both branches. Data are means6 SE

of seven to 10 explants. Different letters indicate statistically signif-icant differences (P , 0.05, by ANOVA). B, Amount of radiolabeledauxin transported basipetally through basal inflorescence stem seg-ments of 36-d-old wild-type, axr1-3, max1-1, and axr1-3 max1-1plants grown on 9 or 1.8 mM NO3

2. Data are means 6 SE of 11 to 12stem segments. There were no statistically significant differences be-tween the two NO3

2 treatments (P. 0.05, by Student’s t test). C, Meanfluorescence of PIN1:GFP at the rootward plasma membrane of xylemparenchyma cells of basal inflorescence stems. Stems were collectedfrom 4-week-old plants harboring a pPIN1::PIN1:GFP transgene andgrown on 9 mM (black bar) or 1.8 mM (gray bar) NO3

2. PIN1:GFPfluorescence at the plasma membrane was assessed using confocalmicroscopy. Data are means 6 SE of 39 to 46 cells. There was no sta-tistically significant difference between the NO3

2 treatments (P . 0.05,by Student’s t test).





SL Deficiency Alters Resource Allocation

To examine the wider consequences of SL deficiencyon low-NO3

2-induced changes in resource allocation,we investigated the effect of N deprivation on therelative allocation of biomass to the root and shoot inthe max1 mutant. The axr1 mutation was not includedin this work because of its highly pleiotropic effects, forexample on fertility (Lincoln et al., 1990), which makethe comparisons difficult to interpret. Both shoot (Fig. 7A)and root (Fig. 7B) biomass were reduced under limit-ing NO3

2 supply, but root biomass was relativelyprotected (Fig. 7C). For the shoot, max1 had slightlyhigher biomass than the wild type under bothN-sufficient and N-deficient conditions, although thiswas not statistically significant on high NO3

2 (Fig. 7A).For the root, while max1 mutants had slightly highermean biomass on high NO3

2, they had slightly lowermean biomass on low NO3

2 (Fig. 7B). Thus, while bothgenotypes showed a shift toward an increased propor-tion of biomass in the root on low NO3

2, in the max1mutant this shift was attenuated (Fig. 7C).

DISCUSSION

Our results clearly show that low NO32 availability

suppresses shoot branching in Arabidopsis. This is

achieved by slowing and early termination of thepostflowering basipetal sequence of bud activationcharacteristic of Arabidopsis plants (Fig. 2). Conse-quently, when NO3

2 is limiting, only the most apicalcauline nodes produce actively elongating branches,whereas all the cauline nodes, as well as some upperrosette nodes, carry active branches when NO3

2 issufficient (Fig. 4). Shoot NO3

2 levels are lower in plantsgrown on low NO3

2 (Fig. 1), but branch suppressionappears to be a response to whole-plant N status ratherthan a direct signaling effect of NO3

2, because a similarresponse was obtained when an alternative N sourcewas used (Fig. 3; Supplemental Fig. S1).

Systemic Signaling and N Status

Both root-to-shoot and shoot-to-root signals are re-quired to explain known responses to whole-plant Nstatus. This is perhaps seen most clearly in split-rootsystem experiments (Zhang et al., 1999; Zhang andForde, 2000; Forde, 2002; Ruffel et al., 2011). Whenboth parts of a divided root system have a low supplyof N, root growth is favored over shoot growth; but

Figure 6. Effects of NO32 supply on the amount of auxin in the PATS.

Amounts of auxin exported from apical (A) or basal (B) stem segments ofwild-type (WT), axr1-3, max1-1, and axr1-3 max1-1 plants grown on 9or 1.8 mM NO3

2 are shown. The inset is an enlargement of the data forthe wild-type stem segments. Eighteen-millimeter apical and basal seg-ments were excised from the inflorescence stems of 6-week-old plants.Data are means6 SE of two to four pools of four stems. Asterisks indicatestatistically significant differences between the two NO3

2 treatments(P , 0.05, by Student’s t test). Different letters indicate statistically sig-nificant differences between the genotypes (P , 0.05, by ANOVA).

Figure 7. Effects of NO32 on resource allocation in SL-deficient mu-

tants. Shoot dry weight (DW; A), root dry weight (B), and root fraction(C) of wild-type (WT) and max1-1 plants grown on 9 or 1.8 mM NO3

2

are shown. Plants were grown for 7 weeks with four plants per pot.Data are means 6 SE of six pots. For both lines, the NO3

2 treatmentsignificantly affected the shoot dry weight, root dry weight, and rootfraction, as indicated by the different letters (P , 0.05, by Student’st test). Asterisks indicate statistically significant differences betweenthe two genotypes (P , 0.05, by Student’s t test).


de Jong et al.




when one part of the root system is in low N and theother part is in high N, root growth on the low-N sideis suppressed. This effect must be mediated via theshoot, since when the shoots are removed the roots canstill respond to local NO3

2 but they have lost the re-sponse to systemic N signaling (Ruffel et al., 2011).

Root-to-Shoot Signals under Limiting N

There are two candidates that can act as N-regulatedroot-to-shoot signals: CK and SL. NO3

2 can up-regulatethe synthesis of CK in roots (Takei et al., 2001;Sakakibara et al., 2006), and this has been implicated inmediating root-to-shoot N signaling (Takei et al.,2002). In Arabidopsis, this response is apparentlymediated by rapid up-regulation of the ATP/ADPISOPENTENYL TRANSFERASE3 (IPT3) CK biosyn-thetic gene (Takei et al., 2004). This gene is up-regulatedby NO3

2 in a cycloheximide-independent manner, andits loss of function causes a severely attenuated CKsynthesis response to NO3

2 addition. However, IPT3transcription is unaffected by other N sources, such asNH4

+; thus, while NO32-induced CK synthesis may

contribute to the branching phenotypes that we ob-served, it is unlikely to be the only cause.Our results demonstrate that SL mutants are com-

promised in their ability to reduce branching when Nis limiting. This result is in contrast with those of Zhuand Kranz (2012), who report that Arabidopsis SL bio-synthetic mutants show the same low branch numberas the wild type when N is limiting. The reason for thisdifference is unclear. Zhu and Kranz (2012) used lowerlevels of N but more rosette branches were retained, soclearly, conditions were different. We consistently androbustly observed high branching on low N for all maxmutants compared with the wild type, indicating thatSL is required for full branch suppression by low N.Furthermore, in a two-bud competition assay, the ef-fects of low N were very similar to SL treatment (Fig. 5A).These results suggest either that there is nearly constitu-tive bud activation in SL mutants or that dynamicchanges in SL levels are involved in mediating theN response. Under this latter hypothesis, a low-Nresponse would trigger the up-regulation of SL syn-thesis. There is mounting evidence that the suppres-sion of Arabidopsis and rice (Oryza sativa) shootbranching by limiting phosphate is mediated in thisway (Umehara et al., 2010; Kohlen et al., 2011). In wild-type plants, phosphate limitation leads to dramaticup-regulation of SL synthesis (Yoneyama et al., 2007b;López-Ráez et al., 2008) and triggers a range of re-sponses, including the suppression of shoot branching.Shoot branching in SL mutants is completely insensi-tive to phosphorus limitation, and the SL pathway isrequired in roots to trigger a range of rapid local re-sponses to phosphorus deprivation (Mayzlish-Gatiet al., 2012). However, the evidence that N limitationhas similar effects is weak. Although the up-regulationof SL biosynthesis by N limitation has been reported,recent results suggest that this might be due to the

effects of N limitation on shoot phosphorus levels(Yoneyama et al., 2012), which in turn affect root SLsynthesis. Similarly, although the ability to reducebranching in response to low NO3

2 seems to requireSL, our results do not support increased SL being amajor cause of reduced branching under N limitation.There is no evidence for the transcriptional up-regulationof SL biosynthetic genes in response to low N supply,and plants grown with limited N supply do not showreduced auxin transport or reduced PIN1 accumulation,which are hallmarks of high SL (Bennett et al., 2006;Lazar and Goodman, 2006; Crawford et al., 2010;Shinohara et al., 2013).

Shoot-Derived Signals under Limiting N

Although we cannot rule out a role for direct effectsof N limitation on either CK or SL synthesis in roots inthe suppression of shoot branching, our results suggesta substantial contribution from systemic shoot-basedeffects. This is consistent with previous evidence forshoot-driven hormonal responses to N (Chen et al.,1998; Walch-Liu et al., 2006; Tamaki and Mercier, 2007;Ruffel et al., 2011). Our data suggest that, under N lim-itation, each active shoot apex exports more auxin,such that the amount of auxin moving in the PATS in

Figure 8. Schematic representation of the hormonal pathways in-volved in the modulation of Arabidopsis shoot branching in responseto N supply. Our results suggest that N limitation increases the amountof auxin exported from each active apex into the PATS of the mainstem. This prevents the activation of more basal branches whilemaintaining the amount of auxin reaching the root. N limitation mayalso affect branching by increasing SL synthesis and decreasing CKsynthesis in roots (dotted lines). Additional feedbacks in the system(not shown here) are also likely to be important; for example, the up-regulation of SL synthesis by auxin is likely important in maintainingplasma membrane PIN1 at constant levels, contributing to auxin ho-meostasis in the system.





wild-type Arabidopsis apical stem segments doubleswhen N is limiting (Fig. 6A, inset). This increase isconsistent with the suppression of shoot branchingconcomitant with the relative protection of root bio-mass observed in response to low N (Figs. 3 and 7).Shoot-to-root auxin transport has previously been im-plicated in the N response. For example, the stimula-tory effects of low N on root growth in maize havebeen associated with increased shoot-derived auxin,and there is some evidence that nodulation dependsboth on high shoot-to-root auxin transport and N lim-itation (Carroll et al., 1985; van Noorden et al., 2006).

The axr1 and max1 mutants both have constitutivelyhigh auxin in the PATS that is unaffected by N supply(Fig. 6). In axr1, this likely results from reduced feed-back inhibition on auxin synthesis due to reducedauxin sensitivity (Romano et al., 1995; Prusinkiewiczet al., 2009). In max1 mutants, it is also likely due toreduced feedback inhibition on auxin synthesis, but inthis case it is caused by increased auxin transport awayfrom the sites of synthesis (Bennett et al., 2006;Prusinkiewicz et al., 2009; Crawford et al., 2010;Shinohara et al., 2013). The additivity of this pheno-type in the double mutant is consistent with differentmechanisms operating in the two mutants. The in-ability of these mutants to change their auxin levels inresponse to low N could contribute to their reducedresponse to N deprivation (Fig. 4A).

The remaining response of each mutant could bemediated by auxin-independent mechanisms, such asreductions in CK mediated directly by N status, or inthe case of axr1-3, increases in SL. In this context, it isinteresting that the shoot elongation suppressionobserved on low N is MAX1 independent but AXR1dependent (Fig. 4B). This suggests that the max1 mu-tant retains some auxin-regulated changes induced bylow N, despite constitutively high auxin levels in thePATS. One possibility is N-induced changes in AXR1-dependent auxin sensitivity, as suggested for roots(Vidal et al., 2010).

Nutrient Responses and Auxin-Mediated Bud Inhibition

Taken together, our data suggest that in wild-typeplants, low N results in increased auxin export fromactive shoot apices, which reduces shoot branchingwhile maintaining strong root growth (Fig. 8). There isa substantial body of evidence that auxin in the mainstem can inhibit bud activation by preventing auxintransport canalization out of buds, thereby preventingtheir growth (Li and Bangerth, 1999; Bennett et al.,2006; Prusinkiewicz et al., 2009; Balla et al., 2011;Shinohara et al., 2013). A rapid primary response to SLsis to reduce PIN1 accumulation at the plasmamembrane,making canalization harder to achieve, thus reducingthe number of buds that can activate (Crawford et al.,2010; Shinohara et al., 2013). As a result, SL mutantshave increased branching, increased auxin transport,and buds that are resistant to auxin-mediated growth

inhibition. Therefore, an important role for SL in branchinhibition on low N is to clamp plasma membrane PIN1at a constant level, thereby rendering buds sensitive toN-mediated increases in apical auxin. The reducedability of max1 mutants to respond to N limitation,therefore, is likely due to their inability to increase budauxin export on low N combined with the general dif-ficulty in preventing the establishment of auxin trans-port canalization out of buds in the absence of SL.

In addition, auxin, signaling through the AXR1-dependent pathway, can up-regulate the transcriptionof SL biosynthetic genes and down-regulate the tran-scription of CK biosynthetic genes (Nordström et al.,2004; Hayward et al., 2009). Both of these hormoneshave been proposed to act locally in buds to regulatebud outgrowth by influencing the transcription ofgenes of the TEOSINTE BRANCHED-CYCLOIDEA-PCF family that are known to be required to inhibitshoot branching (Doebley et al., 1997; Takeda et al.,2003; Kebrom et al., 2006; Aguilar-Martínez et al., 2007;Dun et al., 2012, 2013). This mechanism could con-tribute to branch reduction on low N. Furthermore,increased SL produced in this way is predicted to beimportant for clamping plasma membrane PIN1 levelsdespite auxin-induced increases in PIN1 transcription.However, the failure of the max1 and axr1 mutants toshow an increase in auxin in the PATS on low N leavesthese hypotheses untested.

CONCLUSION

Shoot branching is regulated by the actions and in-teractions of at least three systemically moving planthormones: auxin, SL, and CK. There is an increasingbody of evidence that the environmental control ofshoot branching is mediated at least in part by thishormonal network. Concordantly, our data suggestthat the effects of N supply on branching depend atleast in part on both SL and auxin signaling. ReducedN supply is associated with an increase in the amountof auxin moving in the PATS of apical stem segments(Figs. 6 and 8), which is not observed in the axr1 auxinsignaling mutant or the max1 SL synthesis mutant.This auxin increase can plausibly contribute to thechanges in branching phenotype and root fraction weobserved in response to N supply in the genotypestested. However, the complexity of the feedback in-teractions in the hormonal network make it difficult torule out other mechanisms of N action, and indeed, theremaining N responses in the mutants support theexistence of such additional mechanisms.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Arabidopsis (Arabidopsis thaliana) wild type, accession Columbia, isogenicmutants axr1-3 (Lincoln et al., 1990), max1-1 (Stirnberg et al., 2002), and max2-1(Stirnberg et al., 2002), and transgenic plants harboring the pPIN1::PIN1:GFPtransgene (Benková et al., 2003) were used. Plants were grown in greenhouseconditions with a temperature regime of between 15°C and 24°C. To provide


de Jong et al.



constant long-day conditions (16/8 h of light/dark), natural daylight wassupplemented with artificial light to approximately 150 mmol photons m22 s21.

Plants were grown on low-NO32 substrates consisting of either 90% sand

(Leighton Buzzard sand from WBB Minerals) and 10% (v/v) soil or 50% sandand 50% (v/v) Terragreen (Oil-Dri). These substrates were wetted with Ara-bidopsis salts (ATS) solution (Wilson et al., 1990). High-NO3

2 treatments (9 mM)were supplied as 5 mM KNO3 and 2 mM Ca(NO3)2. For low-NO3

2 treatments(1.8 mM), the 5 mM KNO3 was replaced by 1 mM KNO3 and 4 mM KCl, and2 mM Ca(NO3)2 was replaced by 0.4 mM Ca(NO3)2 and 1.6 mM CaCl2. Nutrientsolutions containing other concentrations of NO3

2 were adjusted accordingly.In the ATS solutions used to study the effect of NH4

+ and NO32, the 5 mM

KNO3 and 2 mM Ca(NO3)2 were replaced by appropriate quantities of NH4Cl,KCl, and CaCl2. From 2 weeks after sowing, the plants were fed on a weeklybasis, using 10 mL of nutrient solution per plant.

To obtain the root and shoot dry weights, plant tissues were dried at 70°Covernight and subsequently redried and cooled on silica gel. The root fractionwas calculated as the dry weight of the roots divided by the total dry weightof the plant.

NO32 Content Analysis

Leaf tissues were collected 4 weeks after germination, just prior to bolting.The tissues were dried and ground to a homogenous fine powder. NO3

2 wasextracted from 5 mg of powder with deionized water for 30 min at 80°C. TheNO3

2 concentration was determined by the rapid colorimetric method de-scribed by Cataldo et al. (1975).

Auxin Analysis

For the auxin export assay, 18-mm segments were excised from the boltingstems of 6-week-old plants. The basal ends of these segments were incubated in50 mL of 2.5 mM diethyldithiocarbamate for 24 h. Four samples of 15 micro-liters (each) were pooled together, and two to four such pools per genotypewere collected. The auxin was purified from the exudate and quantified by gaschromatography-selected reaction monitoring-mass spectrometry as describedby Prusinkiewicz et al. (2009).

The auxin transport assay was performed as described by Bennett et al.(2006) with minor modifications. Fifteen-millimeter basal stem segments wereexcised from the bolting stems of 6-week-old plants. The apical ends of thesesegments were incubated in 30 mL of 0.53 ATS solution, containing 1 mM

14C-labeled indole-3-acetic acid (American Radiolabeled Chemicals). After 24 h ofincubation under constant light conditions, the 5-mm basal ends of the seg-ments were cut and left in 80% (v/v) methanol for 48 h to extract the radio-label. The amount of radiolabel was measured by scintillation in the presenceof MicroScint-20 (PerkinElmer).

Microscopy

Wild-type plants, homozygous for the pPIN1::PIN1:GFP transgene (Benkováet al., 2003), were grown on high and low NO3

2. Four weeks after ger-mination, the basal segments (15 mm) of the primary inflorescence stemswere excised, longitudinally sectioned by hand with a razor blade, and im-mersed in water as described by Shinohara et al. (2013). The sections wereobserved using a Zeiss LSM 780 confocal microscope. Images were acquiredwith excitation at 488 nm and an emission spectrum from 493 to 550 nm. Thefluorescence intensity of PIN1-GFP at the plasma membrane of the xylemparenchyma cells was quantified using Zeiss Zen 2010 software. For eachcondition, regions of four to 11 xylem parenchyma cells were selected foranalysis from each of five stem segments.

Two-Bud Assays

The two-bud assays were performed as described by Ongaro et al. (2008)with minor modifications. Wild-type and max2 mutant plants with boltingstems that carried two cauline nodes with associated buds were selected. Thesize of the buds was not bigger than 2.5 mm. These two-node segments wereexcised and transferred to 1.5-mL microcentrifuge tubes with the appropriateATS solutions containing 1 mM GR24 (LeadGen Labs) or 0.1% (v/v) acetone asa carrier control. To reduce evaporation, tubes were placed in a tray with wetfilter paper on the bottom and a propagator lid. The trays were transferred to a

growth room under the following conditions: 16 h of light, 8 h of dark, tem-peratures of 19°C to 22°C day and 18°C to 20°C night, and light intensity ofapproximately 60 to 100 mmol m22 s21. Bud lengths were measured daily for 7 d,and the ATS solutions in the 1.5-mL microcentrifuge tubes were replenishedwhen necessary. The RGI was calculated as the length of the longest bud di-vided by the total length of both buds.

Jar Assay

To grow plants under sterile conditions, seeds were surface sterilized in10% (v/v) chlorine bleach and then washed with 70% ethanol (v/v, 13) andsterile distilled water (63). After 48 h of stratification at 4°C, seeds were sowninto 500-mL Weck jars containing 50 mL of solid ATS medium (Wilson et al.,1990) with 0.8% (w/v) agar, 1% (w/v) Suc, and, depending on the treatment,3 mM NH4

+ + 3 mM NO32, 6 mM NO3

2, 3 mM NH4+ + 6 mM NO3

2, or 9 mM

NO32. In each jar, six seeds were evenly spaced, and in total, 10 jars per

treatment were used. The jars were placed in a growth room under the followingconditions: 16 h of light, 8 h of dark, temperature of 20°C, and light intensity ofapproximately 100 to 130 mmol m22 s21. Five weeks after germination, branchesof 10 mm or more were counted.

Supplemental Data

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

Supplemental Figure S1. Comparison of the effect of ammonium and ni-trate as N sources on shoot branching under sterile conditions.

Supplemental Figure S2. Branching response of SL-biosynthesis and sig-naling mutants to nitrate supply.

Supplemental Figure S3. Two repeats (A and B) of the experiment pre-sented in Figure 4, showing the mean number of secondary shoots ofauxin-signaling and SL-biosynthesis mutants in response to nitrate sup-ply.

Supplemental Table S1. Transcriptomic changes of SL-biosynthetic andsignaling genes in response to nitrate.

ACKNOWLEDGMENTS

We thank Roger Granbom and Joe Johnson for excellent technical assis-tance.

Received May 5, 2014; accepted July 21, 2014; published July 24, 2014.

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