Inoculation of selenium hyperaccumulator Stanleya pinnata and related non-accumulator Stanleya elata

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1 Physiologi Plntrum Sndinvin Plnt Physiology Soiety, ISSN Inoultion of selenium hyperumultor Stnley pinnt nd relted non-umultor Stnley elt with hyperumultor rhizosphere fungi investigtion of effets on umultion nd speition Stormy Dwn Lindlom, Sirine C. Fkr, Jessi Lndon, Pige Shulz, Ben Try nd Elizeth A. H. Pilon-Smits, Biology Deprtment, Colordo Stte University, Fort Collins, CO, 853, USA Advned Light Soure, Lwrene Berkeley Ntionl Lortory, Berkeley, CA, 97, USA Correspondene *Corresponding uthor, e-mil: Reeived 9 July 1 doi:1.1111/ppl.19 Little is known out how fungi ffet elementl umultion in hyperumultors (HAs). Here, two rhizosphere fungi from selenium () HA Stnley pinnt, Alternri seleniiphil (A1) nd Aspergillus leporis (AS117), were used to inoulte S. pinntnd relted non-ha Stnley elt. Growth nd nd sulfur (S) umultion were nlyzed. Furthermore, X- ry miroproe nlysis ws used to investigte elementl distriution nd speition. Growth of S. pinnt ws not ffeted y inoultion or y. Stnley elt growth ws negtively ffeted y AS117 nd y, ut omintion of oth did not redue growth. lenium trnslotion ws redued in inoulted S. pinnt, nd inoultion redued S trnslotion in oth speies. Root distriution nd speition were not ffeted y inoultion in either speies; oth speies umulted minly (9%) orgni. Sulfur, in ontrst, ws present eqully in orgni nd inorgni forms in S. pinnt roots. Thus, these rhizosphere fungi n ffet growth nd nd/or S umultion, depending on host speies. They generlly enhned root umultion nd redued trnslotion. These effets nnot e ttriuted to ltered plnt speition ut my involve ltered rhizosphere speition, s these fungi re known to produe elementl. Redued trnslotion my e useful in pplitions where toxiity to herivores nd movement of into the food hin is onern. The finding tht fungl inoultion n enhne root umultion my e useful in iofortifition or phytoremedition using root rop speies. Introdution lenium is n essentil element for mny speies inluding humns, ut n e toxi t higher levels. In res where soil levels or iovilility re low, defiieny in livestok is serious nd ostly prolem (Ms et l. 11). In high- res, toxiity n our nd use helth or ehviorl prolems nd even deth in nimls nd humns (Aldosry et l. 1). Although hs not een shown to e essentil to plnts, it hs een reported to e enefiil t low levels, inresing ntioxidnt tivity nd promoting Arevitions μxanes, μ-x-ry sorption ner-edge struture; μxrf, μ-x-ry fluoresene mpping; HA, hyperumultor; MEA, mlt extrt gr; MeCys, methyl-selenoysteine; Cysth, selenoystthionine; Met, selenomethionine. Physiol. Plnt. 13

2 growth (for review, see Pilon-Smits et l. 9). lenium is hemilly similr to sulfur (S) nd redily tken up nd ssimilted y plnts vi the S ssimiltion pthwy (for review, see Terry et l. ). The pity of plnts to umulte from soil is limited y the onentrtion nd iovilility of s well s the physiologil properties of the plnt. Geneti nd environmentl omponents tht ontriute to plnt umultion nd tolerne my e exploited to prevent oth defiieny (vi iofortifition) nd toxiity (vi phytoremedition). Plnt speies differ in their pity to umulte. Most plnts re sensitive to t high levels nd umulte no more thn 1 mg kg 1 DW under nturl onditions (Beth 198). However, smll numer of plnt speies endemi to seleniferous soils n umulte to levels typilly 1-fold higher thn other lol vegettion, rehing 1 15 mg kg 1 DW (Beth et l. 1939). These so-lled hyperumultors (HA)s differ from non-has with respet to their iohemistry nd physiology. Miro-foused X-ry fluoresene (μxrf) mpping nd K-edge X-ry sorption ner-edge struture (XANES) hve shown tht non-ha plnts sequester primrily in the lef vsulture s selente (de Souz et l. 1998, Freemn et l. ), ut HAs umulte predominntly in the lef epidermis in orgni forms with C--C onfigurtion, whih ontin oupled to two orgni groups (Freemn et l. ). HA Astrglus isultus (Fee) showed speifi sequestrtion in the lef trihomes in the form of C--C ompounds tht were identified s methyl-selenoysteine (MeCys) nd γ -glutmyl-mecys using liquid hromtogrphy mss spetrosopy (Pikering et l. 3, Freemn et l. ). HA Stnley pinnt (Brssiee) sequestered in its lef epidermis in speilized ells long the lef mrgin in the form of C--C ompounds tht were identified s 8% MeCys nd % selenoystthionine (Cysth), wheres non-ha Stnley lesens showed diffuse distriution throughout the lef nd umulted 1% Cysth (Freemn et l., 1). The mino id MeCys does not get inorported into proteins, nd n therefore e sfely umulted. This is thought to e the min mehnism of the hypertolerne of HAs (Neuhierl nd Bok 199). While the iohemistry nd physiology of hyperumultion hve reeived lot of ttention, there is still very little known out the plnt miroe intertions of HA plnts or of ny elementl HA (for review, see Alford et l. 1). In generl, plnt miroe intertions n e mutulisti, pthogeni nd/or sprophyti. Sometimes, plnt fungl mutulism n turn pthogeni when the plnt eomes stressed nd/or sprophyti fter the plnt dies, s hs een shown for mny grss symionts in the Alternri genus (Dugn nd Lupien ). Some of the known enefiil effets of miroes on plnts inlude: stimultion of plnt root growth nd hnges in root morphology (de Souz et l. 1998), protetion from pthogens nd herivores s well s from ioti stresses suh s drought, slt, het nd toxi elements (for review, see Hrmon 11). Mny plnts nnot survive without their fungl symiont(s), inditing n essentil role of plnt fungl mutulism in the evolution of plnt stress tolerne (Rodriguez nd Redmn 8). A similr dependene on miroil symionts my hve plyed role in the evolution of hyperumultion. lenium umultion hs likely lso plyed role in the evolution of miroil prtners. There is evidene of wide rnge in tolerne in fungi. In deomposition study onduted on nturlly seleniferous soil, high- lef litter ontined more ulturle miroes thn low- lef litter (Quinn et l. 11). Of the hypotheses presented s to why elementl umultion evolved in plnts, the elementl defense theory, stting tht plnts re proteted y the umulted element from pthogens nd herivores, hs een well supported (for reviews, see Poshenrieder et l., Boyd 7, El Mehdwi nd Pilon-Smits 1). Most elementl defense studies hve foused on plnt herivore intertions, ut one study using Brssi june demonstrted tht plnts treted with were etter proteted ginst two fungl pthogens, n Alternri nd Fusrium speies (Hnson et l. 3). The ojetive of this study ws to investigte whether two rhizosphere fungi from HA S. pinnt n ffet uptke, umultion, speition nd/or growth of S. pinnt s well s of relted non-ha, the -sensitive speies Stnley elt (El Mehdwi et l. 1). The two fungi used in this study were desried erlier y Wngeline nd Reeves (7) nd further hrterized y Lindlom et l. (13). Alternri seleniiphil (A1) nd Aspergillus leporis (AS117), isolted from the rhizoplne of S. pinnt, re oth highly tolernt nd umulte high levels of, minly in elementl form ( ). The pity of these fungi to onvert other, toxi forms of to inert, insolule my not only onfer tolerne to the fungus ut lso ffet their host plnt s uptke, umultion nd tolerne. As mny fungi re promisuous with respet to their host rnge, root symionts of HAs my hve similr effets on non-has. If fungi n indeed ffet umultion nd tolerne in rnge of plnts, they my e useful for iofortifition nd phytoremedition tehnologies. Physiol. Plnt. 13

3 Mterils nd methods Biologil mteril Stnley pinnt seeds were otined from Western Ntive ed, Coldle, CO. Stnley elt seeds were olleted in Southern Nevd y Jennifer Cpp (Colordo Stte University). The two fungl isoltes were otined s desried previously y Wngeline nd Reeves (7), Wngeline et l. (11) from S. pinnt growing on nturlly seleniferous soil round Fort Collins, CO. They re (1) A. seleniiphil (A1) nd () A. leporis (AS117). Fungl ultivtion Fungi were ultivted under ontinuous fluoresent light t C in seled Petri dishes ontining hlfstrength mlt extrt gr (.5 MEA, Difo, Detroit, MI) supplemented with 3 μm sodium selente. Plnt ultivtion The S. pinnt nd S. elt seeds were surfe-sterilized y rinsing for min in % leh, followed y five 1-min rinses in sterile wter. eds were germinted on hlf-strength Murshige nd Skoog medium with 1 g l 1 surose (Murshige nd Skoog 19) under ontinuous light t 3 C in plnt growth inet. After 1 dys the seedlings were refully trnsferred to 1-l ones filled with stem-sterilized grvel (in the ottom) nd orse snd (on top). The plnts were further ultivted in greenhouse under 1 h/8 h L/D photoperiod t 3 C. The plnts were wtered iweekly with qurter-strength Hoglnd s solution (Hoglnd nd Arnon 1938) for weeks nd then inoulted with fungi s desried elow. Inoultion The S. pinnt nd S. elt inoultion tretments onsisted of fungl isoltes A1, AS117 or no inoulum. Twelve replite plnts were used for eh inoulted tretment. Plnts were inoulted with stndrd quntity of fungl hyphe nd/or spores. The fungi were grown on.5 strength MEA pltes for 5 dys, nd fungl mterils were olleted t the perimeter of the fungl olony. In preprtion for plnt inoultion, myeli nd spores or spore hins of A1 were merted in sterile wter in 1.5 ml tues using sterile glss eds nd miropestle. Frgments of hyphe, spores nd spore hins of A1 were quntified using hemoytometer to estimte mm hyphe, spores nd spore hins ml 1 wter. Spores from AS117 were olleted in sterile wter with 1% Tween. A1 nd AS117 were diluted to finl onentrtion of mm hyphe ml 1 wter nd spores mm ml 1 wter, respetively. The inoul were delivered vi pet moss to the plnts in the greenhouse. Eh plnt reeived 1.5 ml of pet moss sturted with 1 ml of inoulum or pet moss with sterile wter for the ontrol tretments. The tretment ws delivered to the plnts vi wtering with 1.5 μm N O twie weekly eginning t the time of inoultion. This onentrtion ws hosen so s to not indue toxiity in the non-ha. After inoultion, plnts nd fungi were oultivted for 13 weeks efore hrvest. At hrvest the plnt roots were gently removed from their ontiners nd snd smples from the rhizosphere olleted for fungl nlysis. The roots were then wshed nd dried for 8h t 5 C for elementl nlysis. Fungl nlysis on snd Rhizosphere snd smples olleted on the dy of hrvest were plted on.5 strength MEA medium. After 3 dys the presene of fungl olonies morphologilly indistinguishle from the inoulum ws verified. Elementl nlysis Smples were digested in nitri id s desried y Zrins et l. (1987). Indutively oupled plsm tomi emission spetrometry ws used to determine elementl onentrtions in the id digest (Fssel 1978). Elementl distriution nd speition X-ry miroproe nlysis ws performed on intt frozen root mteril from S. pinnt nd S. elt tht hd een supplied with 1.5 μm N O. Elementl tissue distriution nd hemil speition were determined using μxrf mpping nd μxanes spetrosopy, respetively, oth s desried y Lindlom et l. (13). Sttistil nlysis The softwre JMP-IN (3.., SAS Institute, Cry, NC) ws used for sttistil dt nlysis. A Student s t-test ws used to ompre differenes etween two mens. Anlysis of vrine followed y post ho Tukey Krmer test ws used when ompring multiple mens. It ws verified tht the ssumptions underlying these tests (norml distriution, equl vrine) were met. Replites tht hd died efore the end of the experiment were not inluded in sttistil nlysis. Physiol. Plnt. 13

4 Dry weight (g) S. pinnt Shoots Roots Dry weight (g) S. elt Shoots 8 Roots Control A1 AS117 Control A1 AS117 Fig. 1. Biomss of Stnley pinnt shoots nd roots (top) nd Stnley elt shoots nd roots (ottom) tht were either uninoulted or inoulted with rhizosphere fungi A1 or AS117. Shown vlues re the men± stndrd error of the men (SEM). Lower se letters ove rs indite sttistilly signifint differenes (P <.5). Results Fungus-inoulted plnts were onfirmed to still hror the originl inoulum, sed on morphologil hrteristis of rhizosphere smples ultured on Petri dishes (not shown). The dry weight iomss of HA S. pinnt ws not ffeted y fungl inoultion, tretment or the omintion of oth (Fig. 1, top; P >.5, two-wy ANOVA). The shoot nd root iomss of the non-umultor S. elt ws negtively ffeted y tretment for the A1-inoulted group, s ws root iomss for the non-inoulted group (Fig. 1, ottom). The root dry weight ws lower for the A. leporis (AS117)- inoulted group thn for the uninoulted ontrol, ut only in the presene of (Fig. 1, right ottom). lenium toxiity, prtiulrly in the lte stges of the experiment, presented itself s die-k from the tips of S. elt leves nd eventully, deth of 5 of the originl 1 uninoulted plnts within the experimentl group. The other tretment groups on verge hd >8% of the originl replites living t the end of the experiment. There ws signifint intertion etween AS117 nd the tretment; individully they redued root iomss ut when omined they did not (Fig. 1, ottom, two-wy ANOVA, P =.1). For the shoots similr pttern ws present ut ws not signifint t the 95% onfidene level (two-wy ANOVA, P =.8). lenium levels in the roots of A1-inoulted S. pinnt were 3% higher ompred with the uninoulted plnts or to plnts inoulted with AS117 (Fig., left). In shoots of inoulted plnts, there were twofold lower levels ompred with uninoulted plnts. As result, the rtio of shoot nd root onentrtion ws twofold to threefold lower in inoulted S. pinnt. As S is hemilly similr to, S tissue onentrtion ws lso determined in the shoots nd roots. Shoot S onentrtion ws lower in inoulted plnts thn in uninoulted plnts, ut only in the presene of (Fig., right). The shoot-to-root S onentrtion rtio in AS117- inoulted plnts ws lower thn in uninoulted plnts; A1-inoulted plnts showed n intermedite rtio. In -treted plnts, root S onentrtion ws signifintly lower in ll ses, wheres in the shoots, S levels were only redued y in fungus-inoulted S. pinnt. The totl mount of umulted, lulted s the produt of onentrtion nd iomss, ws twofold higher in the roots of A1-inoulted S. pinnt ompred with AS117-inoulted or uninoulted plnts (Fig. 3, left). Totl shoot umultion ws 1.5- to -fold lower in plnts inoulted with either fungus. The trnslotion ftor (totl shoot umultion/totl root umultion) ws on verge 5 in uninoulted S. pinnt, i.e. there ws fivefold more in shoots thn roots ompred with only twofold more in shoots thn roots in inoulted plnts. The totl umulted per plnt (root nd shoot) ws 1.5 times lower in AS117-inoulted plnts thn in uninoulted plnts (P <.5); A1-inoulted plnts were intermedite in this respet nd not signifintly different from the other two tretments. In the presene of, there ws no effet Physiol. Plnt. 13

5 µg g 1 DW µg g 1 DW shoots -roots S-shoots S-roots 1 [Shoot] / [Root] S. Control A1 AS117 Control A1 AS117 Fig.. lenium (left) nd S (right) onentrtion in the shoots nd roots, s well s the shoot-to-root onentrtion rtio in uninoulted nd A1- or AS117-inoulted Stnley pinnt. Shown vlues re the men ± SEM. Lower se letters ove rs indite sttistilly signifint differenes (P <.5). of fungl inoultion on shoot nd root totl S umultion (Fig. 3, right). However, in the sene of, totl S umultion in the shoots ofs. pinntws highest in the A1-inoulted plnts nd twofold higher thn in the uninoulted plnts (P <.5). Whole-plnt S ws lso twofold higher in the A1-inoulted plnts ompred to uninoulted S. pinnt without (P <.5); AS117-inoulted plnts showed intermedite results etween A1- nd uninoulted plnts, nd this tretment ws not signifintly different from either other. Tissue levels were not ffeted y fungl inoultion in S. elt (Fig., left). There were lso no differenes in S tissue levels in the presene of (Fig., right). However, in the sene of, root S levels were higher in A1-inoulted plnts ompred with AS117- nd uninoulted plnts, nd shoot S levels were elevted in inoulted plnts ompred with uninoulted plnts. lenium-treted S. elt plnts hd lower S in roots nd shoots in ll ses. The -relted redution in S umultion in the shoots ws more pronouned in the inoulted plnts ( 5%) thn in uninoulted plnts ( %). Stnley elt hd lower shoot levels thn S. pinnt for the non-inoulted tretment. Totl root umultion ws twofold higher in AS117-inoulted plnts ompred with uninoulted plnts (Fig. 5, left, P <.5); A1-inoulted plnts showed intermedite root umultion, whih ws not signifintly different from the other two tretments. Totl shoot umultion ws not ffeted y the fungl inoultions. In the presene of, there were no differenes in shoot or root S umultion etween the inoultion tretments (Fig. 5, right). However, in the -treted group there ws n inoultion effet on S prtitioning etween the roots nd shoots of S. elt. There ws reltively less S in the shoot nd more in the root of AS117-inoulted plnts ompred with uninoulted plnts; the A1-inoulted plnts showed intermedite results (Fig. 5, right, ottom). In the sene of, verge S umultion in shoots nd roots ws higher Physiol. Plnt. 13

6 Shoot / Root Totl umulted (µg) Totl umulted (µg) shoots -roots S-shoots S-roots S Control A1 AS117. Control A1 AS117 Fig. 3. Totl (left) nd S (right) umulted in the shoots nd roots, s well s the shoot/root rtio of shoot nd root umultion in uninoulted, A1- or AS117-inoulted Stnley pinnt plnts. Shown vlues re the men ± SEM. Lower se letters ove rs indite sttistilly signifint differenes (P <.5). in A1-inoulted plnts thn in AS117-inoulted plnts (P <.5); uninoulted plnts showed intermedite S umultion. Totl shoot nd root S umultion ws three to four times higher in the sene of thn in the presene of for A1-inoulted plnts; there ws no suh effet on S umultion in AS117-inoulted or in uninoulted S. elt. XRF mpping ws used to determine the loliztion of nd other elements in inoulted nd uninoulted plnts. lenium distriution nd speition were not ffeted y the fungl inoultions for either of the two Stnley speies. Representtive roots of S. pinnt re shown in Fig.. In the tp root ross setion (left six pnels), ws found throughout ut ws more onentrted in the ortex thn the stele (prtiulrly low signl ws deteted from the xylem). There ws one prtiulrly -rih re in the ortex where lterl root ppers to e emerging from the periyle. lenium did not pper to e very undnt in the periderm, where lium (C) nd iron (Fe) were most onentrted (see overly of, C nd Fe in Fig. ). The distriution of zin (Zn) nd ws strongly orrelted (Person r =.87) s is lso evident from the purple olor in the overly of, C nd Zn (Fig. ). From the side view of the lterl root of S. pinnt (Fig., right six pnels), ws present throughout the root nd not onentrted in ny prtiulr re. Clium nd Fe were onentrted in the periphery nd did not orrelte strongly with loliztion (see overly of, C nd Fe, Fig. ). Similr to the tp root, Zn in the lterl root ololized with (Person r =.88) (see overly of, C nd Zn, Fig. ). Elementl distriution in tp root ross setion of S. elt is shown in the left six pnels of Fig. 7. lenium ws present minly in the ortex. Clium nd Fe, on the other hnd, were present mostly in the periphery, in wht ppers to e the periderm. Zin showed ololiztion with (Person r =.78). Elementl distriution in lterl root of S. elt is shown in the right six pnels of Fig. 7. lenium ws onentrted in disrete strutures tht rn longitudinlly through the lterl root nd my represent vsulr tissues. Clium nd Fe lso showed non-homogeneous distriution, while Zn ws more homogeneously distriuted nd ws ololized with Physiol. Plnt. 13

7 [Shoot] / [Root] µg g 1 DW µg g 1 DW shoots -roots S-shoots S-roots S. Control A1 AS117 Control A1 AS117 Fig.. lenium (left) nd S (right) onentrtion in the shoots nd roots, s well s the shoot-to-root onentrtion rtio in uninoulted, A1- or AS117-inoulted Stnley elt plnts. Shown vlues re the men ± SEM. Lower se letters ove rs indite sttistilly signifint differenes (P <.5). (Person r =.85). lenium lso ololized with Fe in the lterl root of S. elt (Person r =.9). XANES ws used to determine the speition of in the roots of S. pinnt nd S. elt from the different tretments. There were no differenes in speition etween the inoultion tretments (results not shown). The two plnt speies were similr in speition (see Fig. 8A for representtive spetr). In the tp root of S. pinnt s well s S. elt most (95%) ws orgni, with C--C onfigurtion, i.e. MeCys, Cysth or selenomethionine (Met); the reminder of the ws inorgni (selente, selenite or ). It should e noted tht the error mrgin of the liner lest squres omintion fitting is ±1%, so the identifition of the minor frtions is not ertin. In the lterl roots of S. pinnt s well s S. elt there ws reltively less C--C (87%) ompred with the tp roots; the remining, minor frtions of gin were inorgni speies (selente or selenite). As S is hemilly similr to nd supposedly uses the sme trnsporters nd enzymes, S distriution nd speition were determined in tp root of S. pinnt (Fig. 8B). Sulfur XRF mpping showed to e distriuted firly evenly throughout the root (Fig. 8B, inset). Sulfur speition nlysis (XANES) in ortex nd periderm showed mixture of orgni nd inorgni S in roughly equl frtions (Fig. 8B). Disussion The ojetive of this study ws to investigte whether two rhizosphere fungi, A. seleniiphil (A1) nd A. leporis (AS117), isolted from the rhizosphere of HA S. pinnt ffet growth, umultion, loliztion nd/or speition in host plnt S. pinnt, swells in the relted -sensitive, non-ha speies, S. elt. Neither the fungl inoultions nor the ddition of ffeted growth in S. pinnt. There ppered to e n Physiol. Plnt. 13

8 Totl umulted (µg) shoots S-shoots Totl umulted (µg) roots S-roots Shoot / Root S Control A1 AS117. Control A1 AS117 Fig. 5. Totl (left) nd S (right) umulted in the shoots nd roots, s well s the rtio of shoot nd root umultion in uninoulted, A1- or AS117-inoulted Stnley elt. Shown vlues re the men ± SEM. Lower se letters ove rs indite sttistilly signifint differenes (P <.5). inrese in iomss when inoulted with A1, ut this ws not signifint; this effet my hve eome more pronouned if the fungus plnt oultivtion hd een llowed to proeed for longer thn 13 weeks. Stnley elt growth ws negtively ffeted y s well s y AS117 inoultion; these effets were not dditive ut rther ntgonisti. The S. elt AS117-inoulted plnts showed the gretest survivl rte in the presene of, nd this ws the only tretment group where the ddition of did not result in redution of iomss, ut rther the iomss ws slightly inresed. The gretest impt of fungl inoultion on S. pinnt umultion ws in A1-inoulted roots, where there ws two times more ompred with uninoulted roots. In ontrst, AS117-inoulted S. pinntontined 1.5 times less per plnt. As to e expeted, the two fungi ffeted the host plnt properties differently; however, some trends held up for oth fungi. lenium trnslotion in S. pinnt ws lower when inoulted with either fungus, judged from oth tissue onentrtion nd totl umulted. The most interesting result of inoultion in S. elt ws up to twofold higher root umultion in AS117-inoulted plnts. This finding ws somewht surprising euse it ws A1 inoultion tht led to enhned root umultion in S. pinnt, gin inditing tht the funtionl signifine of fungl inoultion is host-dependent. Anlysis of S umultion, hemilly similr to, did show similrities with umultion, ut provided further informtion out the different intertions etween host plnt nd fungus. In S. pinnt, trnslotion of S ws somewht redued. In the non- HA S. elt, fungl inoultion hd more of n impt on S thn on umultion. In the sene of, S. elt inoulted with either fungus hd higher shoot S levels. In the presene of, S trnslotion ws lower in AS117-inoulted plnts nd to lesser extent in A1-inoulted plnts. Chemil speition of ws not ltered y inoultion in roots of S. pinnt or S. elt, Physiol. Plnt. 13

9 C C µm µm Fe CFe Fe CFe Zn CZn Zn CZn Fig.. Miro-XRF mps showing distriution of, C, Fe nd Zn in root ross setion (left six pnels) nd lterl root (right six pnels) of Stnley pinnt. C C 7 µm µm Fe CFe Fe CFe Zn CZn Zn CZn Fig. 7. Miro-XRF mps showing distriution of, C, Fe nd Zn in root ross setion (left six pnels) nd lterl root (right six pnels) of Stnley elt. nd ws found in oth speies to e minly orgni with C--C onfigurtion. Thus, the overll effet of the fungi my e to enhne root umultion nd to redue (nd S) trnslotion to the shoots. The effet ws more pronouned for thn for S in HA S. pinnt, nd more pronouned for S thn for in non-ha S. elt, lthough umultion ws enhned in roots of S. elt inoulted with AS117. The mehnism y whih these fungi ffeted trnslotion in the HA, nd minly S umultion in the non-ha, my e tht these two elements re metolized differently in the Physiol. Plnt. 13

10 Fig. 8. lenium (A) nd sulfur (B) speition s determined y XANES. (A) lenium spetr otined from tproot (stele nd ortex) nd lterl roots of Stnley pinnt nd Stnley elt, in omprison with two stndrds, Met ( C--C ompound) nd selenite (O 3, n inorgni selenoompound). (B) Sulfur spetr otined from four lotions in the ortex nd periderm of the tproot of S. pinnt. Orgni nd inorgni S peks were identified using sulfur stndrds (not shown). Inset shows X-ry fluoresene mp showing S (in white) in the tp root from whih the S XANES spetr were otined. two plnt speies. XANES nlysis reveled tht ws present s C--C ompounds in the HA root s well s in the non-ha root, wheres S ws umulted s roughly equl mixture of orgni nd inorgni S ompounds in S. pinnt. It is fesile tht the fungi ffet trnslotion of orgni selenoompounds differently thn the inorgni nd/or orgni S ompounds. In ddition, the non-ha my hve umulted different form of C--C thn the HA. Erlier, C--C ws found to e the predominnt form of in leves of S. pinnt nd this ws identified s 8% MeCys nd % Cysth (Freemn et l. ) In leves of non-ha S. lesens the ws lso 1% C--C, ut this ws identified s 1% Cysth (Freemn et l. 1). Thus, it is possile tht the form of in S. pinnt nd S. elt roots is tully different, nd if so, different C--C ompounds my e differently ffeted y the fungus. The finding tht lthough similr root distriution ws somewht different etween S. pinnt nd S. elt my suggest tht the ws present in different forms in the two speies. While ws present t the highest levels in the ortex in oth speies, in S. elt ws present more exlusively in the ortex. As the stele ontins the vsulr tissues, the low levels in the stele of S. elt my indite tht there is reltively less in the vsulr ore to e trnsloted. Thus, the somewht different loliztion nd, potentilly, speition of my hve mde the in S. elt less ville for trnslotion. The possile mehnism ehind the oserved redution in nd S trnslotion in the presene of the fungi my e redution of nd S to elementl, insolule nd S y the fungi in the rhizosphere, reduing iovilility nd therefore uptke nd trnslotion. These fungi were isolted from the rhizoplne of S. pinnt, nd thus were in lose ontt with the root surfe, nd oth fungi hve een shown to produe (Lindlom et l. 13). If these fungi hemilly redue (nd S) on the root surfe, it is fesile tht redued, insolule (nd S ) is trpped in fungl hyphe tthed to roots, whih my explin the oserved enhned root nd S levels. The XANES nlysis on inoulted roots did not find sustntil frtion in tproot ross setions or lterl roots, ut the miroem (15 μm ) my hve missed fungl hyphe. In ddition to possile redution of or S, trnslotion of nd S my hve een ffeted y fungl voltiliztion in the rhizosphere, reduing uptke nd trnslotion. AS117 inoultion in S. elt led to the lowest plnt levels, nd AS117 hs een oserved to strongly voltilize (Wngeline 7). The fungus AS117 hd different intertions with the two plnt speies, nd intertion with non-tolernt S. elt ws -dependent. It ppers tht AS117 meliorted the negtive effet of on -sensitive S. elt. The mehnism, s desried ove, my involve redution of in the root zone to inert,or voltiliztion of, oth of whih these fungi hve een demonstrted to e ple of (Lindlom et l. 13). Alterntively, it my e hypothesized tht meliorted the pthogeniity of AS117. If so, the ddition of my hve triggered systemi defense mehnisms in the plnt tht mde it more resistnt to the fungus. Indeed, hs een reported to upregulte the prodution of the hormones sliyli id nd jsmoni id in two Stnley speies (Freemn et l. 1). In plnts treted with selente, root S onentrtion ws lower in oth speies. Shoot S onentrtions were lower in -treted S. elt, ut not in S. pinnt. As selente nd sulfte re nlogous ompounds, the ssoited redution in tissue S levels my e the result of ompetition for the sme trnsporters. The degree of ompetition ws different, however, for the two Stnley speies. In S. pinnt, no redution in totl plnt S umultion ws oserved in the presene of (Fig. 3), wheres the totl S umultion per S. elt plnt ws fourfold redued in the presene of (Fig. 5). Thus, Physiol. Plnt. 13

11 the intertion of nd S ws different in the HA nd the non-ha, perhps owing to differenes in kineti properties of the respetive sulfte/selente trnsporters. The HA ioonentrted to higher levels thn the non- HA, nd in ontrst to the non-ha it did not ompromise S umultion, perhps inditing the presene of seprte selente nd sulfte uptke systems in the HA. Here, it is worth mentioning tht the selente:sulfte rtio ws.5 (1.5 μm selente vs 5 μm sulfte), yet ddition of this reltively low onentrtion of led to fourfold redution in S umultion in S. elt. Either selente strongly outompeted sulfte for uptke or S umultion ws ffeted y seondry toxi effets of the. lenium ololized with Zn in tp roots nd lterl roots of oth Stnley speies, nd in lterl roots of S. elt lso ololized with Fe. The reson for this ololiztion is not ler, ut ould reflet onjugtion y, or inorportion of, these elements in the sme moleules, suh s the heltors glutthione nd phytoheltins. The prodution of these non-protein thiols hs een shown to e indued y vriety of tions nd nions, nd they n ind oth nions (suh s rsenite) nd tions (e.g. Cd + ) (Coett nd Goldsrough ). In onlusion, this study shows tht rhizoplne fungi isolted from HA n enhne root umultion. The effet of inoultion on root umultion ws similr for the HA nd non-ha; however, the speifi fungl inoulnt tht resulted in enhned root umultion ws different for eh Stnley speies. A1 enhned root umultion in S. pinnt, wheres AS117 enhned root umultion in S. elt. The mehnisms ehind the oserved effets of fungl inoultion on umultion re not entirely ler, ut do not pper to inlude ltertion of plnt speition. It is possile tht the fungi lter speition nd iovilility in the rhizosphere. The pprent pity of one fungus (AS117) to llevite redution in iomss from toxiity indites tht speifi fungl inoultion my e useful in pplitions where plnt toxiity is onern. Furthermore, the oserved effet of fungl inoultion in reduing trnslotion to the shoot my e useful in pplitions where shoot umultion is undesirle, e.g. in ses where there is onern for toxiity to herivores nd for the movement of into the food hin. The finding tht fungl inoultion n enhne umultion in roots is lso signifint nd my e useful in root rop iofortifition prties. Aknowledgements We thnk Ami Wngeline for providing the two fungl isoltes nd Jennifer Cpp for olleting nd providing Stnley elt seeds. We lso thnk Jose Rodolfo Vldez Brills for helping with fungl ultivtion nd preprtion. Funding for these studies ws provided y Ntionl Siene Foundtion grnt # IOS to E. A. H. P.-S. The Advned Light Soure is supported y the Offie of Siene, Bsi Energy Sienes nd Division of Mterils Siene of the U.S. Deprtment of Energy (DE-AC-5CH1131). Referenes Aldosry BM, Sutter ME, Shwrtz M, Morgn BW (1) Cse series of selenium toxiity from nutritionl supplement. Clin Toxiol 5: 57 Alford ÉR, Pilon-Smits EAH, Pshke MW (1) Metllophytes view from the rhizosphere. Plnt Soil 337: 33 5 Beth OA (198) The story of selenium in Wyoming. Univ Wyo Agri Exp Stt Bull 77 Beth OA, Gilert CS, Eppson HF (1939) The use of inditor plnts in loting seleniferous soils in the Western United Sttes. I Generl. Am J Bot : 57 9 Boyd RS (7) The defense hypothesis of elementl hyperumultion: sttus, hllenges nd new diretions. Plnt Soil 93: Coett C, Goldsrough P () Phytoheltins nd metllothioneins: roles in hevy metl detoxifition nd homeostsis. Annu Rev Plnt Biol 53: Dugn FM, Lupien SL () Quiesent nd endophyti fungi in grss fmily hosts: mutulists, pthogens, sprophytes nd hithhikers. Proeedings of the Thirty-venth Grss Breeders Work Plnning Conferene, My 1 17, Pullmn, WA, USA El Mehdwi AF, Pilon-Smits EAH (1) Eologil spets of plnt selenium hyperumultion. Plnt Biol 1: 1 1 El Mehdwi AF, Cpp JJ, Fkr SC, lf J, Pilon-Smits EAH (1) Intertions of selenium hyperumultors nd nonumultors during oultivtion on seleniferous or noseleniferous soil the importne of hving good neighors. New Phytol 19: 77 Fssel VA (1978) Quntittive elementl nlysis y plsm emission spetrosopy. Siene : Freemn JL, Zhng LH, Mrus MA, Fkr S, Pilon-Smits EAH () Sptil imging, speition nd quntifition of selenium in the hyperumultor plnts Astrglus isultus nd Stnley pinnt. Plnt Physiol 1: 1 13 Freemn JL, Tmoki M, Stushnoff C, Quinn CF, Cpp JJ, Devonshire J, Fkr S, Mrus MA, MGrth S, Vn Hoewyk D, Pilon-Smits EAH (1) Moleulr mehnisms of selenium tolerne nd hyperumultion in Stnley pinnt. Plnt Physiol 153: Physiol. Plnt. 13

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