Pea nodule gradients explain N nutrition and limited symbiotic fixation in hypernodulating mutants Anne-Sophie Voisin, Marion Prudent, Gérard Duc, Christophe Salon To cite this version: Anne-Sophie Voisin, Marion Prudent, Gérard Duc, Christophe Salon. Pea nodule gradients explain N nutrition and limited symbiotic fixation in hypernodulating mutants. Agronomy for Sustainable Development, Springer Verlag/EDP Sciences/INRA, 2015, 35 (4), pp.0. 10.1007/s13593-015-0328-8. hal-01353750 HAL Id: hal-01353750 https://hal.archives-ouvertes.fr/hal-01353750 Submitted on 12 Aug 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright Agron. Sustain. Dev. (2015) 35:1529–1540 DOI 10.1007/s13593-015-0328-8 RESEARCH ARTICLE Pea nodule gradients explain N nutrition and limited symbiotic fixation in hypernodulating mutants Anne-Sophie Voisin1 & Marion Prudent1 & Gérard Duc1 & Christophe Salon1 Accepted: 17 July 2015 /Published online: 13 August 2015 # INRA and Springer-Verlag France 2015 Abstract Legumes fix atmospheric nitrogen by symbiosis which limits the plant capacity to accumulate N. Furthermore, with soil bacteria in root nodules. Legume yields are limited symbiotic efficiency decreased with increasing nodule num- by the low capacity of N2 fixation. Hypernodulating mutants ber, down to a minimal value for hypernodulating mutants. have been selected decades ago to try to increase nodule num- Thus, to overcome the trade-off between N benefits from N2 ber. However, literature data show that N fixation of fixation and carbon nodulation costs, the hypernodulation trait hypernodulating mutants was not increased compared to pa- should be associated with high shoot growth capacity in rental lines. Here, we study the functional basis of limited N breeding programs. fixation associated to hypernodulation. We grew two wild type genotypes and nine hypernodulating mutants of pea in Keywords Pisum sativum . Hypernodulating mutants . hydroponics in three greenhouse experiments. We measured Nodules . Symbiotic N2 fixation . Symbiosis . Correlation the following traits related to N nutrition during the vegetative networks period: nodule number, plant N uptake, nodule-specific activ- ity, and plant and nodule concentrations. Genetic and environ- mental variations induced nodule gradients. These gradients Abbreviations were used to set quantitative relationships between N nutrition AON Autoregulation of nodulation traits and nodule number. We compared the relationships ob- CCarbon tained for hypernodulating and for wild types. N nutrition NNitrogen traits were analysed together with C nutrition traits, through NNI Nitrogen nutrition index correlation networks. Our results show that higher nodule SNU Specific nitrogen uptake number of hypernodulating mutants is correlated with lower levels of nodule activity, from −25 to −60 %, by comparison to the wild type. Higher nodule number of hypernodulating mu- 1 Introduction tants is also correlated with lower total N uptake by symbiotic − − fixation, from 0to 60 %, by comparison to the wild type. Due to their unique ability to fix atmospheric N2 in symbiosis Findings demonstrate that N nutrition is not a factor limiting with soil bacteria, legumes have a major role to play in sus- growth in hypernodulating mutants, as shown by N nutrition tainable agriculture. As such, they can provide protein-rich index higher than 1, indicating N nutrition in excess. The feed or food in soils poor in nitrogen (N) at low environmental correlations suggest that limited N2 fixation in cost. However, N2 fixation remains a limiting factor of growth hypernodulating mutants arises from restricted shoot growth, and yields, as it is often impaired by biotic and abiotic factors in agricultural conditions (Corre-Hellou and Crozat 2005). This can be partly attributed to the low ability of the N2 fixing * Anne-Sophie Voisin nodules to withstand stresses. Two main reasons can account [email protected] for this particularity of legume plants: first, nodule number is limited by the host plant (see below); second, legume plant 1 INRA, UMR1347 Agroécologie, BP 86510, F-21000 Dijon, France response to restore its N status following a stress mainly relies 1530 A.-S. Voisin et al. on the enhancement of nodule biomass and on the increase of an adjustment to the host plant N requirements, at mini- nodule number, as shown in split-root experiments in which mum carbon cost for nodule formation. In one part of the nodulated root system was deprived of N2 hypernodulating mutants, which are characterised by de- (Jeudy et al. 2010). Indeed, no short-term increase of N2-spe- fective AON regulation, the loss of nodule number regu- cific activity of the nodules still exposed to N2 could be ob- lation occurs simultaneously with loss of nitrate sensitiv- served, whereas this was the case for root-specific nitrate up- ityofnodulation(Oka-KiraandKawaguchi2006). This take in root systems partly deprived of nitrate (Jeudy et al. suggests that the AON pathway shares common elements 2010). Therefore, increasing symbiotic fixation through an with the control of nodule development by the whole increase of nodule number and/or biomass could be a prom- plant N status (Jeudy et al. 2010). Therefore, we hypoth- ising perspective. esized that hypernodulation would first impact the rela- Two major pathways involved in the control of nodule tionships between nodule number and the plant N status, number have been identified. First, an internal regulation leads and that this would in turn impact carbon nutrition traits. to an “auto-regulation of nodule number” (AON) by the host In deregulated mutants, our hypothesis is that excessive plant, through a systemic feedback repression of nodulation nodule formation in hypernodulating mutants could lead by pre-existing nodules (Reid et al. 2011; Mortier et al. 2012 to an imbalance between nodule biomass and nitrogen for reviews). Deregulated mutants with a hypernodulating needs of plants. Nevertheless, only sparse information is phenotype were isolated in several legume species (Novak available on the impact of hypernodulation on nitrogen 2010;Reidetal.2011). In the case of pea (Pisum sativum nutrition in relation with carbon nutrition traits. In a few L.), hypernodulating mutants have been selected after muta- studies, hypernodulation was associated with higher N genesis and three genes have been shown to be involved in the content in root tissues (Day et al. 1986; Novak et al. regulation of nodulation, namely NOD3 (Postma et al. 1988), 2011), in the shoot (Novak et al. 2011 and references SYM29, and SYM28 (Sagan and Duc 1996). On the basis of therein) or in harvested seeds (Bourion et al. 2007), but homologies with Medicago truncatula or Lotus japonicus, this was not related to carbon nutrition traits. In other these three genes have been identified (Krusell et al. 2002, studies, hypernodulation was associated to lower specific 2011;Schnabeletal.2011), NOD3 being of unknown func- N2 fixing activity of nodules (in g N2 fixedpergnodule) tion when the two other genes encode a leucine-rich receptor and high N status as compared to wild type (Voisin et al. like kinase. 2007), without full characterisation of carbon nutrition In an attempt to greatly increase nodule number and there- traits. As such, the actual impact of hypernodulation on fore symbiotic N2 fixation, several hypernodulating mutants N2 fixation remains to be precisely characterised, consid- have been characterised in field or greenhouse studies. The ering both its structural and functional components, and hypernodulating phenotype was never associated to a substan- its relationships with the plant N status and with plant tial increase in N uptake by the mutant plants as compared to carbon nutrition traits, with a special attention to carbon their wild type. Instead, hypernodulation most often limited use for nodule formation. plant growth and productivity (Sagan et al. 1993;Bhatiaetal. In this context, our objectives were to provide a functional 2001;Novak2010; Voisin et al. 2013). Hence, most studies on basis for the low N2 fixation usually associated with hypernodulating mutants have reported negative pleiotropic hypernodulation in pea. Our hypothesis is that there would traits related to carbon nutrition such as depressed shoot be a trade-off between symbiotic N2 fixation and carbon costs growth, shortened internodes or fasciation of the stem, and induced by nodule formation. For this, we used a set of reduction of roots development and growth (e.g., pea: Postma hypernodulating mutants including an allelic series of et al. 1988; Duc and Messager 1989;SaganandDuc1996; SYM29 mutants, thus providing a continuous trend in the Krusell et al. 2002; Bourion et al. 2007;soybean:Matsunami strength of hypernodulation. In a previous study, we have used et al. 2004; Voisin et al. 2013). These pleiotropic effects might this original set of hypernodulating mutants to explain the be a direct effect of the mutated gene on those carbon nutrition pleiotropic depressed growth