Inhibition and recovery of symbiotic N2 fixation by peas (Pisum sativum L.) in response to short-term nitrate exposure

Inhibition and recovery of symbiotic N2 fixation by peas (Pisum sativum L.) in response to short-term nitrate exposure
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  REGULAR ARTICLE Inhibition and recovery of symbiotic N 2  fixation by peas(  Pisum sativum  L.) in response to short-term nitrateexposure Christophe Naudin  &  Guénaëlle Corre-Hellou  &  Anne-Sophie Voisin  & Vincent Oury  &  Christophe Salon  &  Yves Crozat  &  Marie-Hélène Jeuffroy Received: 19 October 2010 /Accepted: 2 May 2011 # Springer Science+Business Media B.V. 2011 Abstract  The design of more sustainable croppingsystems requires increasing N-input from symbiotic N 2 fixation (SNF). However, SNF can be inhibited bynitrate exposure (e.g., soil N-mineralization). Althoughthe effect of nitrate on SNF has been extensivelyinvestigated at the cell scale, few studies havehighlighted the impact of nitrate exposure on nodulenumber and biomass, nodule activity of the SNFapparatus or its ability to recover. Pea plants weregrown in greenhouse conditions in a N-free nutrient solution and exposed to nitrate (5 mM NO 3 − L − 1 ) for one week during either early vegetative growth,flowering or seed filling. After nitrate removal, the plants were grown either under natural light or shade. Nitrate exposure reduced the rate of nodule establish-ment during vegetative growth, whereas it causeddamage to existing nodules when applied during thereproductive stages. Nitrate decreased the specificactivity of nodules regardless of the stage of theexposure. After nitrate removal, an extra wave of nodulation was observed on plants grown under naturallight but only when nitrate exposure occurred beforethe seed filling stage. The recovery of SNF activityafter nitrate removal depended on the amount of carbon available to nodules. Keywords  Pisum sativum  L..SymbioticN 2  fixation. Nodules. Nitrate.Carbon Introduction Owing to the unique process of symbiotic N 2  fixation(SNF), legumes can help to non-renewable resourcesand to avoid environmental impacts related to the useof N (nitrogen) fertilizers (Peoples et al. 1995). Inspite of this tremendous opportunity, during the 20thcentury, cropping area devoted to legumes kept ondecreasing around the world (Crews and Peoples2004). A major explanation is that annual yield of legume crops is more variable than that of non-fixingcrops (Doré et al. 1998; Jensen et al. 2010). Indeed, Plant SoilDOI 10.1007/s11104-011-0817-8Responsible Editor: Katharina Pawlowski.In tribute to Yves Crozat who had taken part in the first  planning of this research project before deceased on 20th June2007.C. Naudin ( * ) : G. Corre-Hellou : V. Oury : Y. Crozat LUNAM Université, Groupe Ecole Supérieured ’ Agriculture, UPSP LEVA (Laboratoire d ’ EcophysiologieVégétale & Agroécologie),55 rue Rabelais, BP 30748, 49007 Angers, Cedex 01,Francee-mail: c.naudin@groupe-esa.comA.-S. Voisin : C. SalonUMR-LEG 102 Génétique et Ecophysiologie desLégumineuses INRA,BP 86510, 21065 Dijon, FranceM.-H. JeuffroyUMR 211 Agronomie INRA AgroParisTech,78850 Thiverval-Grignon, France  complementarity between root assimilation andSNF enables optimal N nutrition (Voisin et al.2002a), but SNF is costly in photosynthates for the plant and often affected by environmental con-straints. As a consequence, yields of legume cropsare lower and less stable than those of other crops.To improve the N use efficiency of legumes andtherefore their applicability in various new croppingsystems less dependent on N inputs, it is important to understand the mechanism underlying their ability to switch rapidly between symbiotic andmineral nutrition in response to heterogeneous andhighly fluctuating soil conditions and crop growthconditions.The level of SNF depends on both symbioticnodule number and biomass, and nodule-specificactivity (i.e., N 2  fixed per g of nodule biomass). SNFis under the control of both long- and short- distanceregulation acting at the plant and at the organ levels.It is also well-known to be modulated by environ-mental factors, including soil mineral N availability(Voisin et al. 2002b). In this regard, numerous studies have established that nitrate ions can inhibit SNF (Streeter  1985a, b). However, how nitrate affect  SNF is still under debate, and several hypothesishave been proposed. At the nodule level, nitrate wasshown to limit nodule appearance either throughmodification of flavonoid production (Bandyopadhyayet al. 1996) or by preventing the infection of root hairs by  rhizobia  (Dazzo and Brill 1978). Concerning nitrate ’ s effect on pre-existing nodules, Streeter andWong (1988) showed that nitrate affects the number  and biomass of nodules more than their activity, byimpairing their growth. At the whole-plant level, nitratehas been shown to reduce nodule activity by changing N composition of the phloem sap, particularly theglutamine content, which is supposed to decreasedioxygen diffusion into the nodule, and subsequentlyto disrupt nodule metabolism and nitrogenase activity( Neo and Layzell 1997). Moreover, a long-term inhibitory effect may also play a major role in theinhibition of N 2  fixation by nitrate through a limitationof the photosynthate flow from shoots to roots(Francisco and Akao 1993). More importantly, the recovery of nodule growthand activity after short-term exposure to nitrate hasnot been extensively investigated. In soybean,Fujikake et al. (2002, 2003) have shown that nodule growth declines in response to short-term nitrateexposure (5 mM) at the beginning of the plant cycle, but rapidly recovers after nitrate removal. Becauseincreasing carbohydrate availability by adding 3%(w/v) sucrose to the medium increased the initialnodule dry weight (DW) (Fujikake et al. 2003), this inhibition effect was suspected to be linked tolimited photosynthate allocation to nodules. Inaddition, N 2  fixation activity has been shown to beenhanced after nitrate removal. Fujikake et al. (2002) hypothesized that early nitrate supply led to an increaseof shoot DW, and thus of increased C supply tonodules, when nitrate inhibition was removed. Indeed,nodule activity largely relies on C supply (Bethlenfalvayand Phillips 1977; Kouchi et al. 1986). Therefore, variations in carbon allocation either between shootsand nodulated roots or between roots and nodules have been suspected to be involved in the plant response tonitrate exposure and in its subsequent reversal. More-over, C allocation to nodules has been shown to bedependent on the phenological stage of the plant. Voisinet al. (2003a) demonstrated that carbohydrate supply to nodules decreases from 45% to 7% of the net  photosynthesis between early vegetative stages and seedfilling. Thus, carbon is mainly allocated downward tonodulated roots during vegetative stages before beingmassively drained to newly appearing C and N sinkssuch as reproductive tissues during the seed filling stage(Jeuffroy and Warembourg 1991; Voisin et al. 2003b). However, studies on short-term inhibition induced by nitrate and subsequent recovery have mainly beencarried out during the first weeks after seedlingemergence; therefore, investigations at later stagesare needed. Jensen (1986) demonstrated that expo- sure of pea plants to nitrate at sowing, first bloom or flat pod stage leads to different effects on plant N-acquisition at maturity, especially in terms of relativecontribution of nitrate absorption and N 2  fixation tototal N uptake. However, the dynamic consequencesof nitrate on nodule number, biomass and activityhave not been investigated. Short-term inhibition of SNF by nitrate in intercropped peas was alsoobserved for a few days after N application in a pea-wheat intercropped field regardless of the stageof N supply ( Naudin et al. 2010). SNF recovery after   N application was observed in intercropped peasonly until pea flowering, whereas SNF was prema-turely abrogated by N fertilization after this stage.These results may indicate effects of nodule age andC availability on the recovery of SNF. Plant Soil  In this context, our main objectives were i) to studythe inhibitory effect of a short-term exposure to nitrateapplied at different plant stages in pea plants, and ii)to analyze the ability of SNF to recover after suchexposure to nitrate with particular attention to Cnutrition. A complete analysis of nitrate ’ s effects onSNF has been carried out to distinguish its effect onthe number, the biomass and the specific activity of fixing nodules under controlled conditions. Material and methods Biological material and growth conditionsAtotalof 1200pea seeds (  Pisum sativum  L. cv Baccara)were weighed, calibrated to 280  –  300 mg and thenseeded for germination on moist filter paper in the darkat 17°C. As soon as the radicle had reached 2  –  3 cm,320 seedlings were selected based on developmentalhomogeneity and transferred to hydroponic culture. Theexperiments were arranged in two randomized complete block designs (one for natural light, one for shading)with four blocs and two replicates per bloc. Plants weregrown in 5.6-L pots covered with a lid with two holesso that each pot hosted two plants. In each pot, an inner wall separated the root systems of the two plants. The pots were covered with aluminum sheeting to maintainthe roots in the dark and to limit excessive heating of the nutrient solution by solar radiation. The solution wascontinuously aerated in each pot compartment by aminimum airflow of 0.25 Lmin − 1 . Plants were inocu-lated with  Rhizobium leguminosarum  bv  vicieae  (strainP221) so as to obtain a concentration of 10 8 cells per  plant. The pH of the nutrient solution was controlledand maintained between 6.5 and 7.5, which enables rhizobia  to efficiently nodulate pea plants (Amarger, personal communication). Nutrient solution (Table 1)was supplied to compensate for plant consumption asnecessary to keep optimal levels in the pots. Thesolution was also completely renewed and re-inoculated at least every two weeks and each time itscomposition was changed. Trace elements and EDTA-FeNa were added to the nutrient solution to avoid anydeficiency (Table 1).After imbibition, germination started on 13/08/08and the first nodules appeared 10 days after seedlinggermination (DAG) (on 23/08/08). The beginning of flowering and the beginning of seed filling occurred42 DAG (on 24/09/08) and 56 DAG (on 08/10/08),respectively. The last harvest was at 78 DAG (on 30/ 10/08) with 40.5%±2.59 of dry matter in grains.During the experiment, pests and diseases werecontrolled with pesticide applications when required.Air and solution temperatures were monitored everyminute by thermocouples and recorded in a data logger (CR10X, Campbell Scientific, Logan, UT, USA). Themean air temperature was 19.6°C±1.9, and the meansolution temperature was 20.6°C±1.9. Cumulative out-door incident photosynthetically active radiation (PAR)above the greenhouse was 940 MJ m − . Due to theseason, the natural photoperiod was decreasing. To reachthe flowering stage, the photoperiod was artificiallyincreased up to 16 h by the use of electric lights (neon).Experimental treatmentsControl plants were both never exposed to nitrate andnever shaded (Table 2). For nitrate treatments, plantswere exposed to a nutrient solution containing nitrate(5 mM NO 3 − L − 1 ) for seven days once during the cropcycle. To be able to discriminate N 2  fixation fromnitrate assimilation, the nitrate supplied was  15  N-labelled K  15  NO 3  (3 atom% 15  N). Three different stages Table 1  Composition of nutrient solutionsSalt concentration (mM L − 1 )Control NMajor nutrient saltsKNO 3  0.00 1.50K  2 HPO 4  0.80 0.80Ca(NO 3 ) 2  0.00 1.75MgSO 4  1.00 1.00CaCl 2  2.50 0.75K2SO 4  0.70 0.08 NaCl 0.20 0.20Minor nutrient saltsCoSO 4  traces tracesH 3 BO 3  3.23 10 − 2 3.23 10 − 2 MnSO 4  6.49 10 − 3 6.49 10 − 3 ZnSO 4  7.65 10 − 4 7.65 10 − 4 (NH 4 ) 6 O 24 Mo 7  1.46 10 − 4 1.46 10 − 4 CuSO 4  3.20 10 − 4 3.20 10 − 4 EDTA-FeNa 5.63 10 − 2 5.63 10 − 2 mean observed atom 15  N (%) 0.00 2.79Plant Soil  of nitrate exposure were tested, during the vegetativestage (N Veg ) (after first nodule appearance), duringFlowering (N Flo ) or at the beginning of Seed Filling(N SF ). After the week of nitrate exposure, half of the plants was grown under natural light (L+) while theother half was grown under shade by using nets untilmaturity (L-). The light level for shaded peas was 65%lower than that for the peas grown under natural light.Harvest and measurementsEach treatment was sampled several times during thecrop cycle: before nitrate exposure, at the end of nitrateexposure and every two weeks until the final harvest.The plants were divided into different organs: initialseeds, nodules, roots, shoots (leaves and stems) andreproductive organs. Before drying, the nodules wereseparated from the roots and placed on a plate to be photographed with a Canon EOS 350D digital (lens:SIGMA 50 mm F2.8 DG). The pictures were analyzedwith ImageJ version 1.40g freeware (National Instituteof Health, USA; http://rsb.info.nih.gov/ij/ ) in order toobtain the number of nodules. The DW of each organwas determined after oven drying at 70°C for 48 h.Roots and nodules were ground together with shoot  parts, and N content was measured according to theDumas procedure (Hansen 1989).  15  N enrichment wasdetermined by mass spectrometry before nitrate expo-sure in the initial seed samples, at the end of nitrateexposure in plant samples and in the nutrient solutions.Calculations and statistical analysisFrom the date t  a   to the date t   b , the quantity of Nderived from air (Ndfa a  →  b ) was calculated as thedifference between total accumulated N (Nplt  a  →  b ) and N derived from nitrate absorption during nitrateexposure (Ndfn a  →  b ): Ndfa a !  b  ¼  Nplt  a !  b    Ndfn a !  b  ð gplt   1 Þ ð 1 Þ  N derived from seeds was not taken into account  because the contribution of seeds to total Naccumulated by the plants did not differ signifi-cantly from the date of the first sampling (14DAG).From the date t  a   to the date t   b  and during the period of nitrate exposure, the amount of absorbednitrate (Ndfn a  →  b ) was calculated as the product of  pea DW times %N content, and the proportion of  plant N derived from nitrate absorption (%Ndfn a  →  b ).%Ndfn a  →  b  was calculated as follows (Rennie andRennie 1983): %Ndfn a !  b  ¼  %A 15  N excess  N    %A 15  N excess  N0 %A 15  N excess Sol    %A 15  N excess  N0 » 100  ð % Þ ð 2 Þ Where:%A 15  N excess  N  isotopic  15  N excess of peas ex <  posed to  15  N-labeled nitrate, at t   b %A 15  N excess  N0  isotopic  15  N excess of peasnever exposed to nitrate, at t   b %A 15  N excess sol  isotopic  15  N excess of the nutrient solution with  15  N-labeled nitrateThe contribution of N derived from air to total Naccumulated in the plant was calculated as the ratio between the quantity of N derived from air and the sumof N derived from absorption and fixation. Thus, fromdate t  a   to date t   b , the contribution of N derived from air  Table 2  Experimental treatmentsReference Date of nitrate exposition (DAG) Dose of nitrate exposition (mM NO 3 − L − 1 ) Growth conditions after nitrate expositionControl  —   0 natural 1ight  N veg  L+ from 14 to 22 5 natural 1ight  N veg  L- from 14 to 22 5 shaded N Flo  L+ from 49 to 57 5 natural 1ight  N Flo  L −  from 49 to 57 5 shaded N SF  L+ from 56 to 64 5 natural light  N SF  L −  from 56 to 64 5 shaded  DAG   days after seedling germination;  Veg   vegetative stage;  Flo  flowering;  SF   seed filling;  L+  and  L − : growth under natural light andunder shade, respectivelyPlant Soil  to total N accumulated in the plant (%Ndfa a  →  b ) wascalculated as follows: %Ndfa a !  b  ¼ ð  Ndfa a !  b = ð  Ndfa a !  b  þ  Ndfn a !  b Þ Þ » 100  ð % Þ : ð 3 Þ From the date t  a   to the date t   b , the specific activity of nodules for N 2  fixation ( ε ) was defined as the ratio between the quantity of fixed N 2  (Ndfa a  →  b ) and theintegrated nodule DW between the date t  a   and the datet   b  (DW nod ).  ε  was calculated as follows (Voisin et al.2007): "  ¼  Ndfa a !  b R  t   b t  a DW nod    dt  ð gN ½ gnodDWd  1   1 Þ ð 4 Þ Where: Z  t   b t  a DW nod    dt   ¼ X  bi ¼ a  ð DW nod ð i Þ  þ  DW nod ð i þ 1 Þ Þ  ð t  ð i þ 1 Þ    t  ð i Þ Þ = 2  ð trapezoid sum approximation Þ  ð 5 Þ Analyses of variance were performed (type IIIsum of squares,  α =5%) and means were comparedusing Tukey ’ s HSD test (Honest Significant Differ-ences,  α =5%) if a main effect or interaction wassignificant using R software (R Development CoreTeam 2009). Results Dynamic of total biomass and N accumulationof control plants and symbiotic N 2  fixation parametersTotal plant dry weight (DW) of peas grown without nitrate steadily increased up to 70 days after germi-nation (DAG) up to 15 gpl − 1 and remained constant afterwards (Fig. 1a). A similar N accumulation patternwas observed until maturity (up to 0.5 gN pl − 1 at maturity; Fig. 1d).Ability of plants to symbiotically fix N 2  wascharacterized via several structural (nodule number and nodule biomass) and functional (nodule specificactivity for N 2  fixation) parameters. The number andDW of nodules increased during the vegetative periodand the early flowering stages (50 DAG) and thenremained constant (Fig. 2a,d). Control plants reachedan average of 658 nodules per plant and 0.26 g DWof nodules per plant at maturity. Nodule specific activitywas defined as the amount of symbiotically fixed N 2  per unit nodule biomass per day. In control plants,nodule specific activity decreased throughout the cropcycle from 0.08 down to 0.02 g N [g nod DW] − 1 d − 1 (Tables 4 and 6). Plant responses to short-term nitrate exposure Total plant growth and N uptake during nitrateexposure Exposure to nitrate for one week at the vegetative stageincreased the rates of both total plant growth and Naccumulation during the exposure (by 45 and 50%,respectively; Table 3). By contrast, total plant growthand N accumulation were not modified during the period of nitrate exposure when it occurred at floweringor seed filling. The contribution of N derived from air to total N-acquisition (%Ndfa) during the exposure tonitrate was very low (from 2 to 12%) regardless of thestage of nitrate exposure (Table 3).  Nodule biomass, number and activity during nitrateexposure When nitrate was supplied at the vegetative stage,the number and DW of nodules increased at alower rate during the period of nitrate exposurecompared to control plants (Fig. 2a,d; Table 4). Similarly to the control plants during the same period, nodule number remained stable duringnitrate exposure when it occurred at flowering andseed filling. However, nitrate exposure during theselate stages decreased nodule biomass whereas thenodule biomass of controls remained stable(Fig. 2b,c and Table 4). Nitrate dramatically decreased nodule specific activity regardless of the stage of exposure (Table 4). The decrease washigher with exposure during seed filling ( − 95%) than Plant Soil
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