Marie-Christine Morère-Le Paven

Quantitative trait loci analysis reveals a correlation between the ratio of sucrose/raffinose family oligosaccharides and seed vigour in Medicago truncatula

Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental conditions. In legumes, raffinose oligosaccharide family (RFO) sugars have been proposed as an easily available energy reserve for seedling establishment. In this study, we investigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determinants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chromosomal regions containing QTL for germination rate or post-germinative radicle growth under optimal or stress conditions co-located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co-located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co-location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds of M. truncatula might negatively affect seed vigour.

Abscisic acid-induced nitric oxide and proline accumulation in independent pathways under water-deficit stress during seedling establishment in Medicago truncatula

Nitric oxide (NO) production and amino acid metabolism modulation, in particular abscisic acid (ABA)-dependent proline accumulation, are stimulated in planta by most abiotic stresses. However, the relationship between NO production and proline accumulation under abiotic stress is still poorly understood, especially in the early phases of plant development. To unravel this question, this work investigated the tight relationship between NO production and proline metabolism under water-deficit stress during seedling establishment. Endogenous nitrate reductase-dependent NO production in Medicago truncatula seedlings increased in a time-dependent manner after short-term water-deficit stress. This water-deficit-induced endogenous NO accumulation was mediated through a ABA-dependent pathway and accompanied by an inhibition of seed germination, a loss of water content, and a decrease in elongation of embryo axes. Interestingly, a treatment with a specific NO scavenger (cPTIO) alleviated these water-deficit detrimental effects. However, the content of total amino acids, in particular glutamate and proline, as well as the expression of genes encoding enzymes of synthesis and degradation of proline were not affected by cPTIO treatment under water-deficit stress. Under normal conditions, exogenous NO donor stimulated neither the expression of P5CS2 nor the proline content, as observed after PEG treatment. These results strongly suggest that the modulation of proline metabolism is independent of NO production under short-term water-deficit stress during seedling establishment.

The Nitrate Transporter MtNPF6.8 (MtNRT1.3) Transports Abscisic Acid and Mediates Nitrate Regulation of Primary Root Growth in Medicago truncatula

A nitrate transporter transports ABA and regulates primary root growth via an ABA-dependent nitrate signaling pathway. Elongation of the primary root during postgermination of Medicago truncatula seedlings is a multigenic trait that is responsive to exogenous nitrate. A quantitative genetic approach suggested the involvement of the nitrate transporter MtNPF6.8 (for Medicago truncatula NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER Family6.8) in the inhibition of primary root elongation by high exogenous nitrate. In this study, the inhibitory effect of nitrate on primary root elongation, via inhibition of elongation of root cortical cells, was abolished in npf6.8 knockdown lines. Accordingly, we propose that MtNPF6.8 mediates nitrate inhibitory effects on primary root growth in M. truncatula. pMtNPF6.8:GUS promoter-reporter gene fusion in Agrobacterium rhizogenes-generated transgenic roots showed the expression of MtNPF6.8 in the pericycle region of primary roots and lateral roots, and in lateral root primordia and tips. MtNPF6.8 expression was insensitive to auxin and was stimulated by abscisic acid (ABA), which restored the inhibitory effect of nitrate in npf6.8 knockdown lines. It is then proposed that ABA acts downstream of MtNPF6.8 in this nitrate signaling pathway. Furthermore, MtNPF6.8 was shown to transport ABA in Xenopus spp. oocytes, suggesting an additional role of MtNPF6.8 in ABA root-to-shoot translocation. 15NO3−-influx experiments showed that only the inducible component of the low-affinity transport system was affected in npf6.8 knockdown lines. This indicates that MtNPF6.8 is a major contributor to the inducible component of the low-affinity transport system. The short-term induction by nitrate of the expression of Nitrate Reductase1 (NR1) and NR2 (genes that encode two nitrate reductase isoforms) was greatly reduced in the npf6.8 knockdown lines, supporting a role of MtNPF6.8 in the primary nitrate response in M. truncatula.

Identification and molecular characterization of Medicago truncatula NRT2 and NAR2 families

Nitrate transporters received little attention to legumes probably because these species are able to adapt to N starvation by developing biological N2 fixation. Still it is important to study nitrate transport systems in legumes because nitrate intervenes as a signal in regulation of nodulation probably through nitrate transporters. The aim of this work is to achieve a molecular characterization of nitrate transporter 2 (NRT2) and NAR2 (NRT3) families to allow further work that would unravel their involvement in nitrate transport and signaling. Browsing the latest version of the Medicago truncatula genome annotation (v4 version) revealed three putative NRT2 members that we have named MtNRT2.1 (Medtr4g057890.1), MtNRT2.2 (Medtr4g057865.1) and MtNRT2.3 (Medtr8g069775.1) and two putative NAR2 members we named MtNAR2.1 (Medtr4g104730.1) and MtNAR2.2 (Medtr4g104700.1). The regulation and the spatial expression profiles of MtNRT2.1, the coincidence of its expression with that of MtNAR2.1 and MtNAR2.2 and the size of the encoded protein with 12 transmembrane (TM) spanning regions strongly support the idea that MtNRT2.1 is a nitrate transporter with a major contribution to the high-affinity transport system (HATS), while a very low level of expression characterized MtNRT2.2. Unlike MtNRT2.1, MtNRT2.3 showed a lower level of expression in the root system but was expressed in the shoots and in the nodules thus suggesting an involvement of the encoded protein in nitrate transport inside the plant and/or in nitrate signaling pathways controlling post-inoculation processes that govern nodule functioning.

Nitrate transporters: an overview in legumes

Main conclusionThe nitrate transporters, belonging to NPF and NRT2 families, play critical roles in nitrate signaling, root growth and nodule development in legumes.Nitrate plays an essential role during plant development as nutrient and also as signal molecule, in both cases working via the activity of nitrate transporters. To date, few studies on NRT2 or NPF nitrate transporters in legumes have been reported, and most of those concern Lotus japonicus and Medicago truncatula. A molecular characterization led to the identification of 4 putative LjNRT2 and 37 putative LjNPF gene sequences in L. japonicus. In M. truncatula, the NRT2 family is composed of 3 putative members. Using the new genome annotation of M. truncatula (Mt4.0), we identified, for this review, 97 putative MtNPF sequences, including 32 new sequences relative to previous studies. Functional characterization has been published for only two MtNPF genes, encoding nitrate transporters of M. truncatula. Both transporters have a role in root system development via abscisic acid signaling: MtNPF6.8 acts as a nitrate sensor during the cell elongation of the primary root, while MtNPF1.7 contributes to the cellular organization of the root tip and nodule formation. An in silico expression study of MtNPF genes confirmed that NPF genes are expressed in nodules, as previously shown for L. japonicus, suggesting a role for the corresponding proteins in nitrate transport, or signal perception in nodules. This review summarizes our knowledge of legume nitrate transporters and discusses new roles for these proteins based on recent discoveries.

Quantitative trait loci analysis reveals a correlation between the ratio of sucrose/raffinose family oligosaccharides and seed vigour in Medicago truncatula: Sugar composition links with seed vigour of M. truncatula

Seed vigour is important for successful establishment and high yield, especially under suboptimal environmental conditions. In legumes, raffinose oligosaccharide family (RFO) sugars have been proposed as an easily available energy reserve for seedling establishment. In this study, we investigated whether the composition or amount of soluble sugars (sucrose and RFO) is part of the genetic determinants of seed vigour of Medicago truncatula using two recombinant inbred line (RIL) populations. Quantitative trait loci (QTL) mapping for germination rate, hypocotyl and radicle growth under water deficit and nutritional stress, seed weight and soluble sugar content was performed using RIL populations LR1 and LR4. Seven of the 12 chromosomal regions containing QTL for germination rate or post-germinative radicle growth under optimal or stress conditions co-located with Suc/RFO QTL. A significant negative correlation was also found between seed vigour traits and Suc/RFO. In addition, one QTL that explained 80% of the variation in the ratio stachyose/verbascose co-located with a stachyose synthase gene whose expression profile in the parental lines could explain the variation in oligosaccharide composition. The correlation and co-location of Suc/RFO ratio with germination and radicle growth QTL suggest that an increased Suc/RFO ratio in seeds of M. truncatula might negatively affect seed vigour.