Nitrogen-Fixing Nodules Are an Important Source of Reduced ...Chrysanthi Kalloniati,a Panagiotis Krompas,a Georgios Karalias,a Michael K. Udvardi,b Heinz Rennenberg,c,d Cornelia Herschbach,c

LARGE-SCALE BIOLOGY ARTICLE

Nitrogen-Fixing Nodules Are an Important Source of ReducedSulfur, Which Triggers Global Changes in Sulfur Metabolism inLotus japonicus

Chrysanthi Kalloniati,a Panagiotis Krompas,a Georgios Karalias,a Michael K. Udvardi,b Heinz Rennenberg,c,d

Cornelia Herschbach,c and Emmanouil Flemetakisa,1

a Department of Biotechnology, Agricultural University of Athens, 11855 Athens, Greeceb Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401c Institute of Forest Sciences, Chair of Tree Physiology, Faculty of Environment and Natural Resources, Albert-Ludwigs-UniversityFreiburg, 79110 Freiburg, GermanydCollege of Science, King Saud University, Riyadh 11451, Saudi Arabia

ORCID IDs: 0000-0003-4846-6435 (G.K.); 0000-0001-6224-2927 (H.R.); 0000-0003-1930-4576 (C.H.)

We combined transcriptomic and biochemical approaches to study rhizobial and plant sulfur (S) metabolism in nitrogen (N) fixingnodules (Fix+) of Lotus japonicus, as well as the link of S-metabolism to symbiotic nitrogen fixation and the effect of nodules onwhole-plant S-partitioning and metabolism. Our data reveal that N-fixing nodules are thiol-rich organs. Their high adenosine59-phosphosulfate reductase activity and strong 35S-flux into cysteine and its metabolites, in combination with thetranscriptional upregulation of several rhizobial and plant genes involved in S-assimilation, highlight the function of nodulesas an important site of S-assimilation. The higher thiol content observed in nonsymbiotic organs of N-fixing plants in comparisonto uninoculated plants could not be attributed to local biosynthesis, indicating that nodules are an important source of reduced Sfor the plant, which triggers whole-plant reprogramming of S-metabolism. Enhanced thiol biosynthesis in nodules and theirimpact on the whole-plant S-economy are dampened in plants nodulated by Fix2 mutant rhizobia, which in most respectsmetabolically resemble uninoculated plants, indicating a strong interdependency between N-fixation and S-assimilation.

INTRODUCTION

Symbiotic nitrogen fixation (SNF) by rhizobia bacteria in legumeplants is a major source of biologically available nitrogen (N) inagricultural and natural ecosystems (Vance, 2001). Rhizobia-legume interaction results in the formation of a specialized plantorgan, the root nodule (Oldroyd et al., 2005), within which thesymbiotic form of rhizobia, called the bacteroid, can reduce at-mospheric N2 to ammonia (NH3). Both partners benefit from thissymbiosis, since the plant is supplied with reduced N and aminoacids from the rhizobia, which in turn receive organic carbonderived from photosynthesis and other nutrients from the plant(Udvardi andDay, 1997; Prell andPoole, 2006;Udvardi andPoole,2013). In N-fixing nodules, plant cells and their microsymbiontsinteract to provide the proper biochemical environment for re-duction and assimilation of N, as well as the efficient exchange ofmetabolites. The establishment of this environment is under-pinned by coordinated changes in gene expression in both theplant and rhizobia, which reprograms many biochemical andmolecular processes in both symbionts (Colebatch et al., 2002,

2004; Fedorova et al., 2002; Ampe et al., 2003; Becker et al., 2004;Kouchi et al., 2004; Küster et al., 2004; Uchiumi et al., 2004;Benedito et al., 2008). Complete elucidation of the physiologicaland biochemical changes and their coordination during noduledevelopment is a major goal of ongoing research.Sulfur (S) is an essential element for SNF, although it has not

been studied much in this context. Nitrogenase, the bacterialenzyme responsible for the reduction of N, is a complex [Fe-S]enzyme (Rees and Howard, 2000) that is produced in relativelyhigh amounts by N-fixing bacteroids (Gaude et al., 2004). NifS isa cysteine desulfurase, which uses L-cysteine for the specificmobilization of S for maturation of nitrogenase, and accumulatesonly under N-fixing conditions (Zheng et al., 1993; Johnson et al.,2005). The Lotus japonicus Sst1 gene, which is expressed in anodule-specific manner, encodes a sulfate transporter that isessential for SNF (Krusell et al., 2005). TheSST1protein appearsto reside on the symbiosome membrane (Wienkoop andSaalbach, 2003) and is thought to transport sulfate from theplant cell cytoplasm to the bacteroids (Krusell et al., 2005). Inlegume nodules, the content of (homo)glutathione, a S-containingantioxidant, correlates strongly with nitrogenase activity and isable to modulate the efficiency of SNF (Dalton et al., 1993;Matamoros et al., 2003; Groten et al., 2005, 2006; El Msehli et al.,2011).In plants and many microorganisms growing with sulfate as

S-source, sulfate is taken up by specific transporters and sub-sequently reduced to sulfide for the biosynthesis of cysteine,

1 Address correspondence to [email protected] author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Chrysanthi Kalloniati([email protected]).www.plantcell.org/cgi/doi/10.1105/tpc.15.00108

The Plant Cell, Vol. 27: 2384–2400, September 2015, www.plantcell.org ã 2015 American Society of Plant Biologists. All rights reserved.

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http://orcid.org/0000-0001-6224-2927

http://orcid.org/0000-0001-6224-2927

http://orcid.org/0000-0003-1930-4576

http://orcid.org/0000-0003-1930-4576

http://orcid.org/0000-0003-1930-4576

http://orcid.org/0000-0003-4846-6435

http://orcid.org/0000-0001-6224-2927

http://orcid.org/0000-0003-1930-4576

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methionine, coenzymes, and iron sulfur clusters of enzymes(Schmidt and Jager, 1992; Brunold, 1993; Leustek and Saito,1999; Höfgen et al., 2001; Kopriva, 2006; Takahashi et al., 2011).The first step of sulfate reduction is its activation to adenosine59-phosphosulfate (APS), catalyzed by ATP sulfurylase (ATPS)(Schmidt, 1972; Brunold, 1993; Leustek and Saito, 1999; Suteret al., 2000). APS is converted to sulfite, SO3

22, by APS reductase(APR) and subsequently reduced to sulfide, S22, by sulfite re-ductase (Suter et al., 2000). Cysteine is formedby incorporation ofS22 into O-acetyl-L-serine (OAS) via O-acetylserine(thiol)lyase.OAS is synthesized from L-serine and acetyl-CoA by serineacetyltransferase. Cysteine serves as the precursor for other re-duced S-compounds, including primary metabolites such asmethionine, GSH, homoglutathione (hGSH), coenzymes, andcofactors, as well as secondary compounds like phytochelatins,glucosinolates, and others (Hassinen et al., 2011; Takahashiet al., 2011; Noctor et al., 2012; Van der Weerden and Anderson,2013).

Previous studies have highlighted the interaction between S-and N-metabolism (Koprivova et al., 2000; Hesse et al., 2004;Kopriva and Rennenberg, 2004). Nodules are the main source ofassimilated N for legumes during SNF. However, little is knownabout the molecular and biochemical mechanisms governingsulfate uptake and assimilation during SNF, either in nodules ornonsymbiotic plant organs. In this article, we present a detailed,whole-plant map of the changes in S-uptake, assimilation, andmetabolism triggered by the establishment of SNF, based ontranscript profiling and biochemical data from L. japonicus. Ourresults highlight the function of N-fixing nodules as a strongsource of assimilated S for the plant and reveal whole-plantreprogramming of S-metabolism upon the establishment ofeffective SNF.

RESULTS

Effects of SNF on S-Metabolite Contents and APR Activity

Changes in whole-plant S-metabolism associated with noduleformation and SNF were studied by comparing sulfate and thiolcontent and APR activity of nodules, roots, stems, and leaves ofL. japonicus plants inoculated with wild-type or DnifA and DnifHmutants of Mesorhizobium loti, and of uninoculated plants. Bothmutant strains of M. loti form ineffective (Fix2) nodules with nonitrogenase activity, in contrast to Fix+ nodules containing wild-type rhizobia. In nodules harboring the DnifA strain, infected cellscontain undifferentiated rhizobia, whereas DnifH nodules containwell-differentiated, albeit ineffective bacteroids (Fotelli et al.,2011).Sulfate levels varied significantly within and between organs,

dependingon thesymbiotic andN-fixingstatusofplants (Figure 1;Supplemental Data Set 1). Sulfate levels were lowest in nodulesof N-fixing plants containing wild-type bacteria and significantlyhigher in nodules containing either of the nifmutants. Sulfate levelswere severalfold higher in roots, stems, and leaves than in nodulesof plants inoculated with wild-type bacteria. Sulfate levels weresignificantly higher in roots of inoculated than of uninoculatedplants, whereas sulfate levels in stems of inoculated plants werelower than of uninoculated plants. Interestingly, sulfate levels ineach organ were highest for nodulated plants that were unable tofix N due to the presence of Fix2 bacteria (Figure 1).APR catalyzes the key step in sulfate reduction (Vauclare et al.,

2002), so the activity of this enzymewasmeasured to gain insightinto changes in this pathway in response to symbiosis. In non-nodulated plants, APR activity was highest in leaves, followed bystems and roots (Figure 1). In N-fixing plants, APR activity was

Figure 1. S-Metabolite Levels and APR Activity in Nodules, Roots, Stems, and Leaves.

Sulfate content (SO422), APR activity, cysteine (Cys), gEC, GSH, and hGSH content in nodules, roots, stems, and leaves of uninoculated L. japonicus

plants and plants inoculated with M. loti wild-type, DnifA, or DnifH strain are shown. Bars represent means (6SE) of five biological replicates. Significantdifferences at P # 0.05 are indicated by different letters.

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highest in leaves, followed by nodules, stems, and roots. In-terestingly, APR activity was lower in leaves and stems of N-fixingplants than in those of non-nodulated plants (Figure 1). APRactivity in nodules appeared to be linked to N-fixation, as bothtypes of Fix2 nodules exhibited very low levels of APR activity.However, the presence of ineffective nodules did not significantlyaffect APR activity in leaves, in comparison to the leaves ofN-fixing plants. These data indicate that N-fixing nodules are animportant site of sulfate reduction in plants.

Consistent with the idea that N-fixing nodules are active insulfate reduction,N-fixingnodulesaccumulated thehighest levelsof thiols, with the exception of hGSH, which was most abundantin leaves (Figure 1). Cysteine content in wild-type nodules was88.46 1.9 nmol g21 fresh weight (FW), almost 5-fold higher thanthat in roots of the same plants. Similarly, N-fixing nodulesexhibited higher g-glutamylcysteine (gEC) contents than rootsinoculated with wild-type M. loti. GSH accumulated almostexclusively in nodules. Nodule GSH content was found to be422 6 15 nmol g21 FW, more than 24-fold higher than the cor-responding roots. hGSH was also abundant in N-fixing nodulesreaching 333 6 53 nmol g21 FW, a value similar to that of thecorresponding roots. In Fix2 nodules formed by both M. loti nifmutant strains, levels of all thiols analyzed were substantiallylower than in N-fixing nodules.

The presence of N-fixing nodules significantly affected thiollevels in nonsymbiotic organs (Figure 1). Roots of N-fixing plantsaccumulated higher levels of all thiols than those of uninoculatedplants. Roots inoculatedwith Fix2 rhizobia contained lower levelsof thiols than roots harboring wild-type rhizobia. The presence ofN-fixing nodules increased the level of gEC, but not other thiols, instems (Figure 1). Nodulation by both nifmutants resulted in loweraccumulation of cysteine, gEC, and hGSH in stems. Thiol accu-mulation, with the exception of GSH, was also higher in leaves ofplants harboring N-fixing nodules than in leaves of uninoculatedplants (Figure 1). Leaves of Fix2 plants accumulated significantlylower levels of cysteine, gEC, and hGSH than leaves of N-fixingplants. Taken together, these results revealed that N-fixingnodules are active in S-reduction and assimilation and possiblyserve as a source of S-metabolites for other organs.

35S-Sulfate Uptake and Distribution within L. japonicusPlants Uninoculated or Inoculated with DifferentM. loti Strains

35S-sulfate was supplied to the root system of intact 6-week-oldplants uninoculated or inoculated with theM. loti wild-type or theDnifHmutant strain. Total 35S-sulfate uptake per plant, uptake perroot system FW, and organ uptake rate were lower in L. japonicusplants inoculatedwithM. lotiwild type than in uninoculated plants(Figures 2and3). Surprisingly, the corresponding values for plantsnodulated by theDnifH strain were in the same range as observedfor uninoculated plants, although the FW of all types of plants(Figure 2) and of their organs (Figure 3) were similar, with theexception of nodules of Fix2 plants. Relative partitioning of 35Sbetween different plant organs was determined by setting total35S-sulfate found within the whole plant to 100%. Relative 35Sabundancewas higher in N-fixing nodules than in ineffective, Fix2

nodules harboring the DnifH strain (Figure 3). The roots of both

types of nodulated plants exhibited lower relative 35S abundancethan uninoculated roots. Thiswas accompanied by higher relative35Sabundance in leavesofplants inoculatedeitherwithM. lotiwildtype or the DnifH strain than in leaves of uninoculated plants(Figure 3).

35S-Flux into the Sulfate, Thiol, and Protein Pools of Rootsand Nodules Exposed to 35S-Sulfate

To further study the potential ofN-fixing nodules asS-assimilators,S-flux through the assimilatory sulfate reduction pathway wasdetermined in nodules and roots of 6-week-old L. japonicus plantsuninoculated or inoculated with M. loti wild-type or DnifH strains.For this purpose, excised nodules and roots were fed with35S-sulfate for 4 h. Sulfate uptake rate and 35S-fluxes into thesulfate, thiol, and protein pools were measured. In addition, totalsulfate and thiol contents as well as APR activity were determined(Figure 4).In N-fixing plants, 35S-sulfate uptake and 35S-flux into internal

sulfate pools was 65% lower in nodules than in roots. However,35S-flux into cysteine in N-fixing nodules was 243 6 28 pmol35S 4 h21g21 FW, representing an almost 4-fold higher flux than inroots of the same plants (Figure 4). This was accompanied bya higher nodule APR activity in N-fixing nodules compared withassociated roots. N-fixing nodules exhibited significantly higher35S-flux into GSH but lower flux into hGSH than did associatedroots. 35S-flux into protein was ;30% lower in N-fixing nodules

Figure 2. Plant FreshWeight and 35S-SulfateUptake ofL. japonicusPlants.

Root systems from intact plants uninoculated and inoculated with M. lotiwild-type and DnifH strain were exposed to 35SO4

22 for 1 h. Plant freshweight, 35S-sulfate uptake per plant, and uptake rate are shown. Meanvalues (6SE) of seven biological replicates are presented. Different lettersindicate significantly different values at P # 0.05.

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than in the associated roots (Figure 4). Fix2 nodules formed by theDnifH strain exhibited lower 35S-sulfate uptake and lower 35S-fluxinto internal sulfate and cysteine pools compared with wild-typenodules. 35S-flux into cysteine in Fix2 nodules was only;10% ofthat in Fix+ nodules and coincided with a 95% reduction in APRactivity in the nonfixing nodules. Likewise, 35S-flux into GSH, hGSH,andproteinwassignificantly lower inFix2 thanFix+nodules (Figure4).

Uptake of 35S-sulfate was lower in roots of plants inoculatedwith wild-type M. loti than in uninoculated roots (Figure 4). Thisresult is consistent with a lower 35S-flux into the internal sulfatepool observed in roots inoculatedwith wild-typeM. loti. However,APRactivitywassimilar in these twotypesof roots, aswas35S-fluxinto cysteine andGSH.On the other hand, 35S-flux into hGSHwashigher and that into protein was lower in roots of N-fixing plantscompared with roots of uninoculated plants. 35S-sulfate uptakewas lower in roots of plants inoculatedwith theDnifH strain than inroots inoculatedwith thewild-type rhizobia,whichwas reflected ina smaller internal 35S-sulfate pool (Figure 4). 35S-flux into cysteineand GSH was slightly lower in Fix2 roots than in roots of Fix+

plants, but similar to roots of uninoculated plants. 35S-flux intohGSHwassubstantially lower (80% lower) in roots inoculatedwiththe M. loti DnifH strain than in roots of Fix+ plants. Also, 35S-fluxinto protein was much lower in roots of Fix2 plants than of Fix+

plants. These results show that effective (Fix+) nodulation shiftssulfate assimilation in roots toward hGSH biosynthesis.

Transcript Profiling of Rhizobial Genes Involved inS-Metabolism during SNF

To assess whether bacteria contribute to nodule S-assimilation,transcript profiling of rhizobial genes involved in S-metabolism

was performed. Relative transcript levels ofM. loti genes involvedin S-metabolism of N-fixing bacterioids and free-living bacteria atexponential and stationary phases of growth are presented inFigure 5 and Supplemental Data Set 1. Changes in relative tran-script level between bacteroids and free-livingM. loti wild type atexponential and stationary phases of growth, and the respectiveP values, are presented in Supplemental Data Sets 2 and 3, re-spectively. The qRT-PCR analyses revealed that expression ofmost of the rhizobial genes involved in S-uptake, reduction, as-similation, and subsequent metabolism were enhanced duringSNF compared with both exponential and stationary phase rhi-zobia. Transcript levels of all sulfate transporters analyzed, exceptCysP2 and CysW, were higher in bacteroids from nodules than into free-living bacteria. The cysA sulfate transporter gene showedthe greatest upregulation compared with exponential (;800-foldchange) andstationary phasebacteria (;300-fold change).Withinthe bacterial sulfate reduction pathway, transcript levels of nodP,coding for the bacterial ATPS, and nodQ, coding for a bifunctionalenzyme with ATPS and APS kinase activity, were significantlyhigher in bacteroids than in free-living bacteria. Relative transcriptlevels of sir, encoding sulfite reductase, were higher in bacteroidsthan in stationary phase rhizobia. Cysteine biosynthesis transcriptlevelswerealsosignificantly higher inbacteroids than in free-livingbacteria. Transcript levels of the cysteine synthase gene, cysK4,which is located in the symbiosis island, a chromosomally in-tegrated element containing all the genes likely to be requiredfor nitrogen fixation (Sullivan et al., 2002), were ;8000-fold and;16,000-fold higher in bacteroids than in exponential- and sta-tionary-phase rhizobia, respectively. Transcript levels for all en-zymes involved in methionine andGSH biosynthesis, except cbl2which was not detected in bacteroids, were the same or higher in

Figure 3. Organ Fresh Weight and 35S-Contents of Nodules and Nonsymbiotic Organs of L. japonicus Plants.

Root systemsof intact plants uninoculated or inoculatedwithM. lotiwild-type orDnifH strainwere exposed to 35SO422. After 1 h, nodules, roots, stems, and

leaves were separated to determine fresh weight and the radioactivity in each organ. Relative organ FWwas calculated from total plant FW (100%). Organuptake rate was calculated from the amount of 35S and the organ FW. The relative 35S-partitioning was calculated from the amount of 35S within the wholeplant (100%). Mean values (6SE) of seven biological replicates are presented. Different letters indicate significantly different values at P # 0.05.


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bacterioids than in free-living rhizobia. Transcript levels for themethionine synthase, metE gene, also located in the symbiosisisland (Sullivan et al., 2002), were more than 4500- and 2000-foldhigher in bacteroids than in exponential- and stationary-phaserhizobia, respectively. Thus, sulfateacquisitionandS-assimilationgenes were strongly upregulated at the transcriptional level inbacteroids compared with free-living rhizobia.

Changes in Transcript Levels of Plant Genes Involved inS-Metabolism during SNF

Transcript levels of plant genes involved in S-metabolism innodules and other organs of nodulated and non-nodulated plantsweremeasured and compared (Figure 6; Supplemental Data Sets1 and 2). Several plant genes involved in S-metabolism weresignificantly upregulated during nodule development, comparedwith roots and other organs (Figure 6). As expected, the nodule-specific sulfate transporter gene Sst1, necessary for SNF (Krusellet al., 2005), was specifically expressed in nodules at all de-velopmental stages. In addition, transcripts of the sulfate trans-porter genes Sultr3.1b and Sultr1.1 were more abundant in

N-fixingnodules,especially inyoungnodulesat14dpostinoculation(dpi), than in other organs. Within the sulfate reduction pathway,Atps was expressed in nodules at all developmental stagesmeasured, although highest mRNA levels were found in roots ofN-fixing plants. Transcripts ofApr2, themainApr isoform in nodules,were 3- to 9-fold higher in nodules at 14 dpi than in nonsymbioticorgans. Sir showed enhanced expression levels in young nodules(14 dpi) compared with other organs. Transcript levels of allcysteine biosynthesis genesweremore abundant in nodules thanin other organs, with the exception of Sat2 whose expressionlevels were lowest in nodules (Figure 6). Sat3 andSat4 transcriptswere found predominantly in nodules. Sat1 and Oastl1 transcriptlevels were enhanced during nodule development in comparisonto the aboveground organs. Oastl3 and Oastl6 transcripts weremost abundant in early stagenodules, althoughOastl6expressionlevels were low compared with the other Oastl isoforms. Allthe remaining Oastl isoforms were consistently upregulated inN-fixing nodules, with Oastl2 and Oastl5 being the predominantOastl isoforms in nodules. Likewise, genes involved in GSHbiosynthesis tended to be more highly expressed in nodules thanin other organs. Transcript accumulation of gecs1, the main

Figure 4. S-Metabolite Content and 35S-Flux into Different Metabolite Pools.

Roots from uninoculated L. japonicus plants and nodules and roots from plants inoculated with wild-type M. loti or with DnifH strain were excised andexposed to 35SO4

22 for 4 h tomeasure sulfate uptake and the 35S-flux into differentmetabolite pools. Sulfate uptake (nmol 35S 4 h21 g21 FW) is indicated onthe graphwith purple axes andAPR activity (nmolmin21 g21 FW) on the graphwith green axes. Sulfate (mmol g21 FW) and thiol content (nmol g21 FW)weredetermined (blue axes). 35S-flux into internal sulfate (nmol 35S4h21g21 FW), thiols (pmol 35S4h21 g21FW), andprotein (pmol 35S4h21g21FW)aregivenongraphs with pink axes. Mean values (6SE) of eight biological replicates are presented. Bars with different letters indicate significant differences (P# 0.05).

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g-glutamylcysteine synthetase isoform since its relative expres-sion levels were over 100-fold higher than gecs2, was highest inroots and in young nodules at 14 dpi. In contrast, transcript levelsof gecs2 were 5- to 10-fold higher in nodules than in roots, re-gardless of the nodule developmental stage. Transcripts of glu-tathione synthetase Gshs were 4- to 8-fold more abundant innodules at all developmental stages than in nonsymbiotic organs.In contrast, relative expression levels of homoglutathione syn-thetase Hgshswere highest in roots and in early stage nodules at14 dpi. In summary, most of the plant genes involved in sulfatetransport and assimilation weremore highly expressed in nodulesthan in other organs during SNF.

Profiling of Bacterial and Plant Transcripts Involved inS-Metabolism in Fix2 Nodules Formed by DnifH andDnifA Strains

Todeterminewhether the transcriptional changesassociatedwithS-metabolism in nodules are linked to active N-fixation, transcriptprofiling was also performed on Fix2 nodules formed by eitherDnifA or DnifH rhizobia. Relative transcript levels of M. loti andL. japonicusgenes in ineffectiveandN-fixingnodulesarepresentedinFigure7 andSupplementalDataSets1, 2 and3.Focusingfirst onbacterial gene expression, transcripts of the cysA sulfate trans-porter were ;50% less abundant in Fix2 nodules formed by theDnifA and DnifH strains than in Fix+ nodules containing wild-type

rhizobia, while transcript levels of the sulfate transporters cysP1,cysU, cysW, st1, and st2 were higher in Fix2 nodules than in Fix+

nodules (Figure 7). However, the relative expression levels of thesetransporters were low in comparison to cysA. Transcript levels ofthe cysteine synthase gene, cysK4, encoding the main isoformexpressed in N-fixing bacteroids, were approx. 85-fold lower in themutantbacteria. The relative transcript levelsofg-glutamylcysteinesynthetase, gshA, were also lower in nodules harboring DnifA andDnifH rhizobia. In both types of Fix2 nodules, transcript levels ofmethionine synthetasemetH and of methionine synthetasemetE,whichwas found tobehighly upregulated inN-fixing nodules,weremuch lower in comparison to N-fixing nodules. In summary, manyof the rhizobial genes required for sulfate uptake and assimilationwere expressedat lower levels inFix2nodules than inFix+ nodules.Most of the plant genes involved in sulfate uptake, reduction,

assimilation, and metabolism were found to be expressed atsignificantly lower levels in Fix2 nodules than in Fix+ nodules(Figure 7; Supplemental Data Sets 1 and 2). This included allsulfate transporter genes, except for Sultr1.3. For the nodule-specificSst1 transporter, transcript levelsweresubstantially lowerin nodules formed by DnifA and DnifH strains compared with thewild-type strain. For the plant sulfate reduction pathway, Atpstranscript levels were lower in nodules harboring DnifH rhizobia,whileApr2andSir transcript levelswere lower inboth typesofFix2

nodules than inFix+nodules. Transcript levelsofSat1,Sat4, andof

Figure 5. Transcript Profiling of M. loti Genes Involved in Sulfate Uptakeand Metabolism during SNF.

Relative transcript levels ofM. loti genes in nodules formed byM. lotiwild-type and free-living rhizobia at the exponential and stationary growthphases.Measurementswere conductedon threebiological replicates, andtranscript levels were normalized using polyribonucleotide nucleotidyl-transferase-encoding transcripts as reference. Mean values (6SE) arepresented in a base e logarithmic scale.

Figure 6. Transcript Profiling of L. japonicus Genes Involved in SulfateUptake and Metabolism during SNF.

Relative transcript levels of L. japonicus genes in nodules formed byM. lotiwild-type strain at various stages of development, namely at 14, 21, and 28dpi, aswell as in nonsymbiotic organsof the inoculated L. japonicusplants.Measurements were conducted on three biological replicates and tran-script levels were normalized using ubiquitin-encoding transcripts asreference. Mean values (6SE) are presented in a base e logarithmic scale.


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Figure 7. Heat Map of Transcriptional Changes in L. japonicus andM. loti Genes Involved in Nodule Sulfate Uptake, Reduction, and Assimilation duringSNF.

(A)Heatmap of fold changes in relative transcript levels of L. japonicus genes in nodules formed byM. lotiwild-type in comparison to the transcript levels inroots of the same plants.(B) and (C)Heatmap of fold changes in relative transcript levels of L. japonicus genes in Fix2 nodules formed by eitherDnifA orDnifH strain, respectively, incomparison to the transcript levels in nodules formed by the wild-type M. loti.(D)Heatmapof fold changes in relative transcript levels ofM. lotigenes inN-fixing nodules in comparison to the transcript levels in free-living rhizobia at theexponential growth phase.(E) and (F)Heatmap of fold changes in relative transcript levels ofM. loti genes in Fix2 nodules formed byDnifA orDnifH strain, respectively, in comparisonto the transcript levels in N-fixing nodules. Measurements were conducted on three biological replicates, and transcript levels of bacterial and plant geneswere normalized using polyribonucleotide nucleotidyltransferase- and ubiquitin-encoding transcripts as references, respectively. Statistically significantdifferences are indicated by asterisks. The whole data set of fold changes and the statistical analysis are available in Supplemental Data Sets 2and 3, respectively.

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all the Oastl isoforms were significantly lower in both Fix2 than inFix+ nodules. Beyond cysteine biosynthesis, mRNA levels ofgecs1 and Hgshs, responsible for gEC and hGSH biosynthesis,respectively, were lower in both Fix2 than Fix+ nodules. The ex-pression level ofGshs, responsible for theGSHbiosynthesis, waslower in the nodules formed by theDnifA strain. The expression ofother plantgenes remainedunaffectedbymutations in the rhizobialnif genes (Supplemental Data Sets 2 and 3).

SNF Triggers Changes in Expression of Plant S-MetabolismGenes at the Whole-Plant Level

To explore the impact that sulfate assimilation in N-fixing noduleshas on whole-plant S-metabolism, we measured and comparedtranscript levels of S-transport and assimilation genes in variousorgans of Fix+ and Fix2 plants. Several plant genes involved inS-metabolism were expressed at lower levels in uninoculatedroots and roots of Fix2 plants than in roots of Fix+ plants, while nogenes were expressed at higher levels in roots of non N-fixingplants (Figure 8). Expression of the sulfate transporter geneSultr1.1 was lower in uninoculated roots and roots of Fix2 plantsthan in roots of Fix+plants. Systemic transcriptional changeswerealso observed in the sulfate reduction pathway. Transcript levelsof Apr1 in uninoculated roots and of Apr2 in roots of Fix2 plantswere lower than in roots of Fix+ plants. Within the cysteine bio-synthesis pathway, transcript accumulation of Sat1,Sat2,Oastl1,Oastl3, and Oastl5 was significantly lower in uninoculated rootsand roots of Fix2 plants than in roots of Fix+ plants. Genes en-coding three additionalOastl isoforms,Oastl2,Oastl4, andOastl6,were also expressed at lower levels in uninoculated roots than inroots of N-fixing plants. Transcript levels of gecs1 were lower inuninoculated roots and roots of Fix2 plants than in roots of Fix+

plants. Finally, mRNA levels of Hgshs were lower in uninoculatedrootsand roots inoculatedwith theDnifHstrain than in rootsofFix+

plants inoculated with the wild-type M. loti.In stems, mRNA levels of the sulfate transporter Sultr3.4awere

lower in uninoculated and Fix2 plants than in Fix+ plants. Tran-script levels of the sulfate transportersSultr1.3 andSultr3.1bwerealso lower in stems of uninoculated plants than in N-fixing plants.In contrast, transcripts of Sultr3.3a were higher in stems of Fix2

than of Fix+ plants. In stems of uninoculated plants, lower tran-script levels were found for all of the genes of the sulfate reductionpathway (Atps,Apr1,Apr2, andSir) and for several genes involvedin cysteine and GSH and hGSH [(h)GSH] biosynthesis, includingSat1, Sat2, Sat4, Oastl1, Oastl4, Oastl5, and gecs1, comparedwith stems of Fix+ plants. Likewise, expression of Apr2, Sat4, andOastl3was lower in thestemsofFix2comparedwithFix+plants. Incontrast, stems of Fix2 plants had higher transcript levels ofSat1,Oastl2, and Oastl5 than did stems of Fix+ plants.

In leaves, transcript levels of the sulfate transporter geneSultr3.3a were lower in uninoculated and Fix2 plants than in Fix+.Transcript levels of the mitochondrial serine acetyltransferasegeneSat4and thecytosolicO-acetylserine(thiol)lyasegeneOastl2were higher in leaves of uninoculated and Fix2 plants than inleaves of N-fixing plants. Transcript levels of Hgshswere lower inleavesofuninoculatedandFix2plants than in leavesofFix+plants.

In summary, the presence of N-fixing nodules with substantialsulfate assimilation capacity had a significant impact on the

expression of many genes involved in sulfate transport and as-similation throughout theplant. Ingeneral, genes involved insulfateuptake were found to be upregulated, while numerous genes forsulfate reduction and assimilation were expressed at lower levelsin other (non-nodule) organs of N-fixing plants than in those ofuninoculated or Fix2 plants (Figure 8). In other words, N-fixationand sulfate assimilation in nodules was accompanied by tran-scriptional reprogramming of many of the plant genes involved inS-assimilation in other organs.

DISCUSSION

It is well established that uptake, assimilation, and metabolism ofN and S are tightly connected in plants (Smith, 1980; Clarksonet al., 1989; Koprivova et al., 2000; Ho and Saito, 2001; Vauclareet al., 2002; Hesse et al., 2004; Kopriva and Rennenberg, 2004;Davidian and Kopriva, 2010). In this context, several studiesdemonstrated the importance of sulfate uptake and metabolismfor theestablishment and functionofSNF (Matamoros et al., 1999,2003; Abbas et al., 2002; Harrison et al., 2005; Krusell et al., 2005;Bianucci et al., 2008; Muglia et al., 2008; Chang et al., 2009;Becana et al., 2010; El Msehli et al., 2011; Frendo et al., 2013).However, little is known about the molecular and biochemicalmechanisms governing S-reduction and metabolism during SNFand the impact of nodulation on the whole-plant S-metabolism.Thisstudy revealed thatnodulesareastrongsourceofassimilatedS for the whole plant and trigger whole-plant reprogramming ofS-partitioning and metabolism.

N-Fixing Nodules Are a Strong Source of Assimilated S

Our results from 35S-flux analysis, APR activity, and S-metabolitemeasurements combined with transcriptomic analysis in nodulesclearly indicate that S-uptake, assimilation, and metabolism areenhanced in both symbionts during SNF. To test whether theenhancement of S-metabolism in nodules is linked to active SNForwhether it is just related to noduledevelopment,weexperimentedwithM. lotiDnifA andDnifHmutant strains. TheDnifA strain triggersaberrant nodule development, fails to differentiate into bacteroids,and has no detectable nitrogenase activity, whereas theDnifH straintriggers normal nodule development but with no detectable nitro-genase activity (Fotelli et al., 2011). The results from these experi-ments with Fix2 nodules revealed that enhanced S-assimilationand metabolism is strongly linked to active SNF. The major stepsof S-uptake and metabolism upregulated in nodules and reg-ulated by SNF are summarized in Figure 9.The first clear hint that N-fixing nodules are an important site of

S-assimilation in L. japonicus came from the observation that theyare themain site of cysteine accumulation in these plants (Figure 1).This observation was consistent with those of Matamoros et al.(1999),who reported thatnodules frompea (Pisumsativum), broadbean (Vicia faba), alfalfa (Medicago sativa), soybean (Glycinemax),mung bean (Vigna radiata), and cowpea (Vigna unguiculata) plantsare also rich in cysteine. In addition, our data suggest that cysteineis synthesized in situ in N-fixing nodules and not imported fromnonsymbiotic organs. This conclusion is supported by the ob-servation that N-fixing nodules appeared to be, together withleaves, a major site of sulfate reduction and assimilation,


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exhibitinghighAPRactivityand35S-flux intocysteine (Figures1, 4,and9). ThehighAPRactivity andcysteine content in noduleswerefound to be linked to active SNF, as APR activity and 35S-flux intocysteine were largely abolished in Fix2 nodules, resulting in sig-nificantly lower cysteine content than in Fix+ nodules. The link

between S-assimilation and active SNF in nodules presumablyreflects the regulation of sulfate assimilation by N-availability(Clarkson et al., 1989; Kopriva and Rennenberg, 2004; Davidianand Kopriva, 2010). Both ammonium and amino acid levels havebeen implicated as significant factors in the regulation of APR

Figure 8. Heat Map of Transcriptional Changes in L. japonicus Genes Involved in Sulfate Uptake, Reduction, and Assimilation at the Whole-Plant Levelduring SNF.

Heat map of fold changes in relative transcript levels in nonsymbiotic organs, including roots (R), stems (S), and leaves (L) of uninoculated plants (A) andplants inoculatedwithM. lotiDnifA (B) andDnifH (C) strains in comparison to the transcript levels in plants inoculated with wild-typeM. loti. Measurementswere conducted in three biological replicates, and transcript levels were normalized using ubiquitin as a housekeeping gene. Statistically significantdifferences are indicated by asterisks. The whole data set of fold changes and the statistical analysis are available in Supplemental Data Sets 2 and 3,respectively.

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activity and subsequent S-assimilation (Brunold and Suter, 1984;Koprivova et al., 2000; Davidian and Kopriva, 2010). In line withthese reports, our data suggest that nodules function as strongsulfate assimilators only in the presence of active SNF, indicatingthat the immediate products of N-fixation act as positive regu-lators of S-assimilation and metabolism in nodules.

Active cysteine biosynthesis in nodules was underpinned bySNF-dependent transcriptional upregulation of many bacterialand plant genes for S-uptake and assimilation (Figure 9). Asa starting point, several plant and rhizobial sulfate transportergeneswere found tobe inducedduring nodule development, in anSNF-dependent manner (Figures 7 and 9). These included theplantgenesSst1andSultr1;1. SST1 is locatedon thesymbiosome

membrane of L. japonicus nodules (Wienkoop and Saalbach,2003) and is believed to transport sulfate from the host cell cy-toplasm to the bacteroids (Krusell et al., 2005). SST1 is crucial forSNF in L. japonicus, as sst1mutant plants develop Fix2 nodules,which indicates that sulfate is a primary source of S for bacteroids(Krusell et al., 2005). Transcriptional upregulation of the rhizobialcysA gene, which encodes one of the five subunits of a sulfatetransport complex (Laudenbach and Grossman, 1991; Kredich,1996; Piłsyk and Paszewski, 2009), may ensure sulfate uptakefrom the periplasmic space into the bacteroids. In Gram-negativebacteria, CysA is known to form a dimeric periplasmic bridgebetween the outer membrane and cytoplasmic membrane sub-units of the sulfate transport complex (Scheffel et al., 2005; Piłsyk

Figure 9. Schematic Presentation of Sulfate Uptake, Reduction, and Assimilation at the Whole-Plant Level during SNF Indicating That N-Fixing NodulesRepresent aSignificantSourceofAssimilatedS for thePlant andTriggerWhole-PlantReprogrammingofS-Metabolismupon theEstablishment of EffectiveSNF.

(A)Differences inS-uptake, reduction, andassimilationobserved inN-fixingplants comparedwithuninoculatedplantsarepresented.NonsymbioticorgansofN-fixingplants are comparedwith the respective organsof uninoculated plants. Nodules are comparedwith roots ofN-fixing plants andbacteroids to thefree-living rhizobia.(B)Differences in S-uptake, reduction, and assimilation observed in Fix2 plants comparedwith N-fixing plants are presented. Nonsymbiotic organs of Fix2

plants arecomparedwith the respectiveorgansofN-fixingplants. Fix2nodulesarecomparedwithN-fixingnodulesandFix2 rhizobia toN-fixingbacteroids.Differences in organ 35S-uptake rate, APR activity, 35S-flux into S-metabolites, sulfate and thiols content, and transcriptional regulation are indicated bypurple, green, orange, blue, and black arrows, respectively.


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and Paszewski, 2009). In the bacteria Salmonella typhimurium,Escherichia coli (Kredich, 1992), and Klebsiella aerogenes (Lynchet al., 1994), activation of genes involved in sulfate uptake, acti-vation, reduction, and assimilation is dependent on high levelsof acetylserine, linking S-assimilation to N availability. Severalbacterial and plant genes involved in cysteine biosynthesis werealso found to be transcriptionally upregulated in nodules in re-sponse toSNF.TheM. loticysteinesynthasegenecysK4, residingon the symbiosis island of the genome (Sullivan et al., 2002), wasstrongly induced in N-fixing bacteroids, but not in Fix2 nodulesharboring rhizobial nifmutants (Figures 5, 7, and 9).Most plantSatand Oastl genes were also induced during nodule developmentin an SNF-dependent manner, including mitochondrial Sat4 andOastl5, and cytosolic Oastl2 and Oastl4 (Figures 6 and 9). InArabidopsis thaliana,mitochondrial serine acetyltransferaseplaysthe predominant role in cellular OAS formation (Heeg et al., 2008;Haas et al., 2008; Watanabe et al., 2008; Krueger et al., 2009;Takahashi et al., 2011; Rennenberg andHerschbach, 2014), whilethe main site of cysteine biosynthesis is considered to be thecytosol (Heeg et al., 2008; Watanabe et al., 2008; Krueger et al.,2009; Rennenberg and Herschbach, 2014). All of these tran-scriptional changes that promote cysteine biosynthesis werelargely abolished in Fix2 nodules (Figures 7 and 9), indicating thatSNF positively affects this process in nodules. Downregulation ofcysteine biosynthesis in Fix2 nodules was reflected in the rela-tively high levels of sulfate that accumulated in ineffective nodules(Figures 1 and 9).

Cysteine is a precursor of other important S-compounds innodules, including g-glutamylcysteine, (h)GSH, and methionine,production of which was also reduced in Fix2 compared with Fix+

nodules (Figures1,4,and9).Biosynthesisof these thiolsappearedto be transcriptionally regulated since several of the rhizobial andplant genes involved were upregulated in N-fixing nodules, incontrast to Fix2nodules (Figures 5, 6, 7, and 9). The importance ofthese thiols in legume-rhizobia symbiosis is well documented. (h)GSH plays a crucial role in nodule development and SNF effi-ciency, since Mt-gECS RNAi-silenced lines of M. truncatula de-veloped smaller nodules with decreased (h)GSH content andlower acetylene reduction activity per nodule than control plants(El Msehli et al., 2011). With respect to methionine, previousstudies have demonstrated that allSinorhizobiummelilotiRmd20Imethionine auxotrophs (metA/metZ, metE, and metF mutants)formed ineffective nodules on alfalfa plants, a phenotype relatedmainly to the reduced number of infected nodule cells and in-complete bacterial differentiation into bacteroids (Abbas et al.,2002). ThisFix2phenotypewascompletely revertedbyadditionofmethionine to the plant nutrient medium, suggesting that infectedplant cells can but normally do not provide sufficient methioninefor bacteroiddevelopment andSNF.Although relevant literature isnot available forM. loti, our results indicate thatM. loti bacteroidsare geared to produce methionine via induction of genes formethionine biosynthesis. These include metE, a gene located inthe M. loti symbiosis island (Sullivan et al., 2002), whose ex-pression was several thousand-fold induced in N-fixing bacte-roids (Figures 5 and 9). Themajority of bacterial genomes encodemore than one enzymatic mechanism for the methylation of ho-mocysteine to generate methionine. However, methionine pro-duction is dependent not only on the genetic/metabolic potential

of the bacteria, but also on the availability of the precursors foreach pathway (Barra et al., 2006). In M. loti, the contribution ofalternative pathways of methionine biosynthesis in free-livingbacteria and N-fixing bacteroids could differ due to differences inthe availability ofmethionine precursors acting asmethyl-donors,namely, 5-methyltetrahydropteroyl-tri-L-glutamate for the co-balamin-independent MetE and 5-methyltetrahydrofolate for thecobalamin-dependent MetH (Mordukhova and Pan, 2013). Fur-ther studies are needed in order to elucidate the role ofmethioninein N-fixing bacteroids, and the construction ofM. loti methionineauxotrophs is underway.Overall, our data suggest that sulfate reduction, assimilation,

and metabolism are activated in N-fixing nodules, although 35S-sulfate uptake was lower in nodules than in roots (Figure 9). Thisresult might reflect the specialized role of roots in sulfate uptakefrom the nutrient medium. Irrespective of lower sulfate uptakecompared with roots, N-fixing nodules exhibited significantlyhigher APR activity and fluxes into cysteine and GSH comparedwith roots.Surprisingly, thiswasobserveddespite thehighnodulethiol content (Figures 1, 4, and 9). This is in contrast to previousstudies where an excess of thiols repressed APR activity andS-assimilation through reduction of 35S-flux into thiols (Herschbachand Rennenberg, 1994; Zhao et al., 1999; Vauclare et al., 2002;Hesse et al., 2004). This apparent discrepancy could reflect dif-ferent biochemical mechanisms regulating S-uptake and assimila-tion and/or compartmentalization of thiol biosynthesis and thiolaccumulation within the infected nodule cells. N-fixing bacteroidscouldactivelyparticipate in the increasedefficiencyofS-assimilationin nodules by the compartmentalization of S-metabolism in theinfected cell. Thus, they appear to be significant players in nodulesulfate assimilation. Most of the rhizobial genes for sulfate uptakeandmetabolism were induced in N-fixing nodules compared witheither free-livingbacteria or Fix2nodules (Figures5and7),makingthem potential sinks for sulfate and sources of thiols. Bacteroidsare able to maintain oxidative phosphorylation to supply energyfor N-fixation (De Visser and Lambers, 1983), despite the very lowfree-oxygen concentrations that are aprerequisite for nitrogenaseactivity (Bergersen, 1982; Kuzma et al., 1993; Udvardi and Poole,2013), via the involvement of leghemoglobin (Appleby, 1984; Ottet al., 2005). Thus, they are well equipped to supply energy forS-assimilation under the microaerobic conditions of nodules.Although more experiments are needed to determine the relativecontribution of N-fixing bacteroids to nodule sulfate assimilationand metabolism, our data indicate that they play a significant rolein this regard.

S-Assimilation in N-Fixing Nodules Influences Whole-PlantS-Partitioning and Metabolism

AlthoughS isacrucial element inmanymolecules involved inSNF,such as proteins and protein cofactors (Zheng et al., 1993; DosSantos et al., 2004; Johnson et al., 2005), our results suggest thatthiols synthesized in nodules do not only fulfill the needs of SNF,but also contribute to theS-economyof thewhole plant (Figure 9).N-fixing nodules, as a likely source of reduced-S, triggered globalreprogramming of sulfate uptake, reduction, and assimilation atboth the transcriptional andmetabolite levels (Supplemental DataSets 1 and 2). Interestingly,many of these changes are dependent

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on SNF because they were not observed in Fix2 plants, which inmost respects resembled, metabolically, uninoculated plants.

Our findings indicate that thiols synthesized in N-fixing nodulescould be subjected to long-distance transport contributing to theS-budget of other organs. Indeed, reduced S-compounds havebeen detected in the xylem sap of several species (Rennenberget al., 1994a, 1994b;Schneider et al., 1994b, 1994a; Schuppet al.,1991; Herschbach andRennenberg, 2001a, 2001b). Compositionand contents of S-compounds in the xylem sap vary betweenspecies, season, S-nutrition, developmental stage, part of thetrunk, aswell aswith the presence ofmycorrhization (Herschbachand Rennenberg, 2001a, 2001b; Herschbach et al., 2012). GSH isconsidered to be the predominant reduced-S compound sub-jected to long-distance transport inplants (Schneideret al., 1994a,1994b; Herschbach and Rennenberg, 1995; Herschbach et al.,2012; Gigolashvili and Kopriva, 2014). However, many plants alsocontain cysteine andgEC in appreciable amounts in the xylemsap(Schupp et al., 1991; Rennenberg et al., 1994a, 1994b; Blaschkeet al., 1996). In xylem sap of beech trees, cysteine is the pre-dominant low molecular weight thiol, whereas GSH is present inminute amounts (Schupp et al., 1991; Rennenberg et al., 1994a;Herschbach et al., 2012). It has been well documented thattransport of thiols in the xylem sap contributes to the whole plantsulfur cycling and nutrition (Rennenberg and Herschbach, 2014).

The higher cysteine content found in leaves of N-fixing plants incomparison to uninoculated and Fix2 plants is unlikely to haveresulted fromenhanced localbiosynthesisbecauseAPRactivity inleaves of N-fixing plants was found to be significantly lower thanin uninoculated plants (Figures 1 and 9). Interestingly, transcriptlevels of both Apr genes in leaves were not affected by thepresence of either nodules or of SNF. Thus, the decrease of APRactivity in nodulated plants may reflect posttranscriptional orposttranslational regulation of APR, which is known to occur inplants (Bick et al., 2001; Kopriva and Koprivova, 2004; Koprivovaet al., 2008; Davidian and Kopriva, 2010; Takahashi et al., 2011).The lowAPRactivityandcysteine levels in the leavesofFix2plantscould account for both the accumulation of sulfate and the lowerthiol levels throughout theplant.Asimilar observationwasmade inArabidopsis, where reducedAPRactivity correlatedwith high foliarsulfate levels (Loudet et al., 2007; Davidian and Kopriva, 2010).

Downregulation of cysteine biosynthesis in the leaves of N-fixing plantswas also apparent at the transcriptional level. Severalgenes involved in this process, including mitochondrial Sat4 andcytoplasmic Oastl2, exhibited significantly lower transcript levelsin leaves of N-fixing plants than of uninoculated and Fix2 plants(Figures8and9). Thehigher cysteine content found in rootsof Fix+

plants compared with uninoculated and Fix2 plants also points tonodules as a source of cysteine for other organs, since root APRactivity and 35S-flux into cysteine were similar in uninoculated,Fix+, and Fix2 plants (Figures 1, 4, and 9).

Our data indicate that GSH may also be transported from theN-fixing nodules to the adjacent roots. Similar Gshs transcriptlevels and35S-flux into theGSHpool in rootsof uninoculated, Fix+,and Fix2 plants do not suggest higher GSH biosynthesis in rootsof Fix+ plants and cannot account for their higher GSH content(Figures 1, 4, 8, and 9). On the other hand, N-fixing nodules ap-peared tobe themainsiteofGSHbiosynthesis, exhibiting relativelyhigh transcript levels of Gshs and 35S-flux into GSH (Figures 4, 6,

and 9). Supporting the notion that nodules are a source of GSH forroots and other organs, GSH is known to be transported over longdistances in plants (Schneider et al., 1994a, 1994b; Herschbachand Rennenberg, 1995; Herschbach et al., 2012; Gigolashvili andKopriva,2014). Incontrast, our resultsdonotpoint to long-distancetransport of hGSH from nodules to other organs, as the elevatedlevelsofhGSHinrootsand leavesofN-fixingplants,comparedwithuninoculated and Fix2 plants, correlated well with upregulation ofthe Hgshs gene in both organs (Figures 1, 8, and 9). Furthermore,greater 35S-flux intohGSH in the roots of Fix+ plants comparedwithuninoculated and Fix2 plants (Figures 4 and 9) is indicative of thelocal biosynthesis of hGSH.The intriguing hypothesis that nodules serve as a substantial

source of thiols for other organs is further supported by the rel-atively low 35S-flux into protein observed in nodules comparedwith roots of Fix+ plants (Figure 4). This indicates that the bio-synthesis of nodule S-containing proteins, including nitrogenasesubunits, generates a lower demand for S-flux into protein thanprotein biosynthesis in roots. Thus, the demands for proteinbiosynthesis in N-fixing nodules cannot account for the highlevels of S-assimilation and cysteine biosynthesis.Previous studies have shown repression of sulfate uptake by an

excess of cysteine or other reduced S-compounds (Herschbachand Rennenberg, 1994; Zhao et al., 1999; Vauclare et al., 2002;Hesse et al., 2004). Such feedback repression could account forthe significantly lower total 35S-sulfate uptake per plant, sulfateuptake per root system FW, and organ uptake rate in Fix+ com-pared with uninoculated and Fix2 plants (Figures 2, 3, and 9).In conclusion, our data show that in the L. japonicus-M. loti

symbiosis, nodules are not only the main site of plant N-assimi-lation but are also a major site of S-assimilation and thiol bio-synthesis. Furthermore, the data indicate that nodules may bea significant source of assimilated S for other organs; certainly, thepresence of Fix+ nodules leads to reprogramming of S-metabolismgenes and altered S-metabolism and S-partitioning throughoutthe plant. The impact of nodules on whole-plant S-economy isdampened in theabsenceof activeSNF innodules, a situation thatforces the plant to switch back to its nonsymbiotic N- and S-assimilation patterns. Future work, employing both plant andrhizobial mutants, will help to clarify the relative contribution ofrhizobia and plant cells to nodule sulfate assimilation and thecontribution of nodules to the overall plant S-metabolism.

METHODS

Plant Material, Bacterial Strains, Culture Conditions, andPlant Growth

Mesorhizobium lotiwild-type strain R7A and the Fix2mutant strains DnifAand DnifH were kindly provided by Clive Ronson (University of Otago,Otago, New Zealand). Both mutant strains form ineffective small whitenodules with no nitrogenase activity (Fotelli et al., 2011). The main dif-ference between the two types of ineffective nodules is thatDnifH nodulescontain well differentiated, although ineffective, bacteroids, in contrast tonodules harboring DnifA strain where infected cells are colonized by un-differentiated rhizobia. All strainsweregrown for 72hat 28°Consolid yeastmannitol broth supplemented with the appropriate antibiotic (30 mg/mLkanamycin for DnifA; 50 mg/mL gentamycin for DnifH). For the liquidcultures, rhizobial minimal medium was used (per 1 liter: 0.25 g


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MgSO4$7H2O, 0.1 g CaCl2$2H2O, 1 g K2HPO4, 0.53 g NH4Cl, 1 g KH2PO4,10 mg FeCl3$6H2O, 20 mg CoCl2, 20 mg Na2MoO4, 20 mg MnSO4$H2O,20mgZnSO4$7H2O, 20mgCuSO4$5H2O, 10 g glucose, 20mgbiotin, 20mgthiamine, and 20 mg vitamin B5).

Lotus japonicus (Gifu B-129) seeds were kindly provided by JensStougaard (University of Aarhus, Aarhus, Denmark). The seeds werescarified for5minwithH2SO4,sterilized for20min in2%(v/v)NaOCl-0.02%(v/v) Tween 20, pregerminated at 18°C in the dark for 72 h, and spot-inoculated with a suspension culture of the respective M. loti strain at anOD600 of 0.1. For each independent experiment, sets of plants were grownatan18-h-day/6-h-night cycle, a22°Cday/18°Cnight temperature regime,and 70% relative air humidity (Handberg and Stougaard, 1992). Un-inoculated plants were watered with nitrate containing Hoagland solution(Hoagland and Arnon, 1950), plants harboring effective nodules werewatered with N-free Hoagland solution, and plants harboring ineffectivenodules were watered alternately with N-free and nitrate-containingHoagland solution.

Thiol Analysis

A total of 30 to 100 mg homogenized frozen plant material was sus-pended in 0.75 mL 0.1 N HCl containing 50 mg of insoluble poly-vinylpolypyrrolidone. The samples were centrifuged for 30 min at 14,000gand 4°C. For reduction of thiols, 120 mL of the supernatant was added to180 mL 200 mM CHES buffer, pH 9.3, and 30 mL 15 mM DTT. After in-cubation at room temperature for 60min, thiolswere derivatized in the darkafter adding 20 mL 30 mM monobromobimane for 15 min. Bimane de-rivatives were stabilized with 250 mL 10% acetic acid and separated withaHPLCsystem (BeckmanGoldSystem), creating agradientwith increasingmethanol on aC-18column (ODS-Hypersil 2503 4.6mm i.d., 5-mmparticlesize; Bischoff Chromotography). Thiol derivatives were determined byfluorescence detection with a Shimadzu RF-535-Fluorescence HPLCdetector (Shimadzu Europe) and quantified by external standards(Schupp and Rennenberg, 1988).

Sulfate Analysis

Fifty milligrams of homogenized frozen plant material was suspended in1mLofdistilledwatercontaining100mgof insolublepolyvinylpolypyrrolidoneto remove phenolic compounds. After shaking for 1 h at 4°C, samples wereboiled for 15 min and centrifuged for 15 min at 14,000g and 4°C. The clearsupernatant was used for sulfate analysis by anion exchange chromatog-raphy. Anions were separated on an IonPac column (AS9-SC, 2503 4 mm;Dionex) elutedwithamixtureof 2mMNa2CO3and0.75mMNaHCO3ataflowrate of 0.5mLmin21. Sulfatewasdetectedby a conductivity detectormodule(CDM; Dionex).

APR Activity and Protein Determination

APSreductaseactivitywasmeasuredasproductionof 35S-sulfite, assayedas acid volatile radioactivity, formed in the presence of 75 mM AP35S and4 mM dithioerythritol, according to Brunold and Suter (1990).

Radioactivity in proteins was determined in 30 mg of frozen samples.Powdered plant samples were suspended in 1 mL of extraction buffer(125mMTris-HCl, pH6.8, and6%SDS) by vigorous vortexing.Proteinwasprecipitated in 10% trichloroacetic acid for 30 min on ice, washed, andredissolved in 0.2 M NaOH. The amount of 35S incorporated into proteinwas determined in the redissolved protein by liquid scintillation countingafter adding 5 mL of liquid scintillation fluid (HiSafe 2; Perkin-Elmer).

35S-Sulfate Uptake of Intact L. japonicus Plants

Root systemsof intact 6-week-oldL. japonicusplantswere transferred into2mLN-freeHoaglandsolution adjusted to0.1mMSO4

2–containing10mCi

(3.73 105 Bq) of carrier-free 35SO422 (Hartmann Analytic). Incubation was

performed at room temperature (20 to 25°C) and 180 6 30 µE (Osram L58W/12 Lumilux de luxe Daylight and Osram L 58W/77 Fluora). After 1 h,sulfate uptakewas terminated bywashing the root system three timeswithnonradioactive N-free Hoagland solution. Nodules, roots, stems, andleaves were separated, weighed, frozen in liquid N, and stored at 220°Cuntil analysis of 35S.

35S-Sulfate Flux Analyses

35S-flux analyses were conducted according to Scheerer et al. (2010).Nodules or roots were excised from 6-week-old L. japonicus plants. Theexcised organs were then transferred into 10mLN-free Hoagland solutionadjusted to 0.1 mM SO4

2– and supplemented with 3% sucrose containing150mCi (5.53106Bq) of carrier-free 35SO4

22 (HartmannAnalytic). After 4 hof incubation at room temperature, sulfate uptake was stopped bywashing rootsornodules three timeswithnonradioactiveN-freeHoaglandsolution. Samples were frozen and powdered in liquid N and stored at–20°C until analysis of the 35S-content and the 35S-flux into sulfate, thiols,and protein.

35S Analyses

The 35S-content of the samples was determined by measurement of ra-dioactivity in 20 mg of each sample, powdered under liquid N. After solu-bilizationwith a tissue solubilizer (Soluene 350; Perkin-Elmer), the dissolvedsamples were bleached with 200mL of H2O2 (30%) overnight. After adding5 mL of scintillation fluid (HiSafe 2; Perkin-Elmer), radioactivity was de-termined by scintillation counting (Wallac System 1409) (Herschbach andRennenberg, 1996).

35S-Metabolite Analyses

Sulfate was quantified by anion exchange chromatography and specificradioactivityof thesulfatewasdeterminedby liquid scintillation countingof1.2-mL fractions of the eluate after anion exchange chromatography (seeabove). The amount of 35S in thiolswasdetermined in theeluate afterHPLCanalysis (see above). A total of 4 mL of scintillation fluid (HiSafe 3; Perkin-Elmer) was added to each 1-mL fraction, and radioactivity was determinedby liquid scintillation counting. Radioactivity in thiols of the differentfractionswascorrelated to the respective peaksafter fluorescent detection(Scheerer et al., 2010).

Transcript Analysis Using qRT-PCR

Total RNA was isolated from free-living bacteria harvested during expo-nential and stationary growth, aswell as nodules and nonsymbiotic organsof L. japonicus plants either uninoculated or inoculated with M. loti wild-type, DnifA, or DnifH stains according to Brusslan and Tobin (1992) andquantified by spectrophotometry and agarose gel electrophoresis. RNAwas treated with DNase I (Promega) at 37°C for 45 min to eliminatecontaminating genomic DNA. First-strand bacterial or plant cDNA wasreverse transcribed using Super-Script II (Invitrogen) and random hex-anucleotides or oligo(dT)12–18mer (Invitrogen) primers, respectively. qRT-PCR was performed on the Stratagene MX3005P using iTaq fast SYBRGreen Supermix with ROX (Bio-Rad Laboratories) and gene-specific pri-mers.Detailed information for all genes in this study, includingnames,genesymbols, accession numbers, predicted topology, and gene-specificprimers, is presented in Supplemental Data Set 4. PCR cycling started at95°C for 10min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.The primer specificity and the formation of primer dimers were monitoredby dissociation curves and agarose gel electrophoresis on a 4% (w/v) gel.The expression levels of the M. loti polyribonucleotide nucleotidyl-transferasegene (mlr5562) andaL. japonicusubiquitin genewere usedas

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internal standards for the bacterial and plant cDNAs, respectively. Rel-ative transcript levels of the gene of interest (X) were calculated as a ratioto the internal standard gene transcripts (H), as (1+E)–DCt, where DCt wascalculated as (Ct

X–CtH). PCR efficiency (E) for each amplicon was cal-

culated by employing the linear regression method on the log (fluores-cence) per cycle number data, using LinRegPCR software (Ramakerset al., 2003).

Statistical Analysis

All statistical analyses were performed using SigmaStat 3.5. Plannedcomparisons were performed by applying pairwise t tests. When thenormality test failed, the Mann-Whitney Rank Sum test was used. Sta-tistical significance was postulated for P # 0.05. The statistical analysiswas performed on the values from independent plant sets used as bi-ological replicates within the same organ, while wild-type nodules werealso comparedwith the inoculated roots. For sulfate and thiol content, APRactivity, and 35S-flux analysis, two independent experiments were per-formed at different time periods under controlled conditions, includingcontrols and all genotypes, comprising independent plant sets used asbiological replicates. Transcript analysis was performed on three in-dependently grown plant sets and bacterial cultures used as biologicalreplicates.

Accession Numbers

The accession numbers of the sequences used in this article are providedin Supplemental Data Set 4.

Supplemental Data

Supplemental Data Set 1. Relative transcript levels of genes involvedin S-metabolism.

Supplemental Data Set 2. Fold changes in transcript levels of genesinvolved in S-metabolism.

Supplemental Data Set 3. Statistical analysis of results presented inSupplemental Data Set 2.

Supplemental Data Set 4. List of genes involved in S-metabolism.

ACKNOWLEDGMENTS

We thank Ursula Scheerer for technical assistance provided duringexperiments employing 35S and Susanne Mult for preparing 35S-APS.This work was funded by a joint DAAD and Greek State ScholarshipFoundation project.

AUTHOR CONTRIBUTIONS

E.F., H.R., C.H., and C.K. designed the research. C.K. performed theexperiments and analyzed the data. P.K. and G.K. performed the experi-ments for the bacterial gene expression analysis. C.K., P.K., and G.K.carried out bioinformatics analyses. C.K., E.F., C.H., H.R., and M.K.U.contributed to experimental design and interpretation. C.K. and E.F. wrotethe article. C.H., H.R., and M.K.U. critically reviewed the article andcontributed to the preparation of the final version.

Received February 5, 2015; revised July 20, 2015; accepted August 3,2015; published August 21, 2015.

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