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BMC Plant Biol
2019 Aug 30;191:380. doi: 10.1186/s12870-019-1978-5.
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Disruption of the Lotus japonicus transporter LjNPF2.9 increases shoot biomass and nitrate content without affecting symbiotic performances.
Sol S
,
Valkov VT
,
Rogato A
,
Noguero M
,
Gargiulo L
,
Mele G
,
Lacombe B
,
Chiurazzi M
.
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BACKGROUND: After uptake from soil into the root tissue, distribution and allocation of nitrate throughout the whole plant body, is a critical step of nitrogen use efficiency (NUE) and for modulation of plant growth in response to various environmental conditions. In legume plants nitrate distribution is also important for the regulation of the nodulation process that allows to fix atmospheric N (N2) through the symbiotic interaction with rhizobia (symbiotic nitrogen fixation, SNF).
RESULTS: Here we report the functional characterization of the Lotus japonicus gene LjNPF2.9, which is expressed mainly in the root vascular structures, a key localization for the control of nitrate allocation throughout the plant body. LjNPF2.9 expression in Xenopus laevis oocytes induces 15NO3 accumulation indicating that it functions as a nitrate importer. The phenotypic characterization of three independent knock out mutants indicates an increased shoot biomass in the mutant backgrounds. This phenotype is associated to an increased/decreased nitrate content detected in the shoots/roots. Furthermore, our analysis indicates that the accumulation of nitrate in the shoot does not affect the nodulation and N-Fixation capacities of the knock out mutants.
CONCLUSIONS: This study shows that LjNPF2.9 plays a crucial role in the downward transport of nitrate to roots, occurring likely through a xylem-to-phloem loading-mediated activity. The increase of the shoot biomass and nitrate accumulation might represent a relevant phenotype in the perspective of an improved NUE and this is further reinforced in legume plants by the reported lack of effects on the SNF efficiency.
20105XLAXM Italian Ministry of Education (Progetti di Rilevanza Nazionale), PRIN 2010/2011, PROROOT, CUP B25C13000290007 Rete delle Biotecnologie in Campania, Progetto Bio Industrial Processes - BIP, ANR-14-CE34-0007-01-HONIT Agence Nationale de la Recherche, F/050005/00/X32 Italian Ministery of the Economic Development (MISE)
Fig. 1. LjNPF2.9 expression in different organs. RNAs are extracted by wild type plants grown on B5-Gamborg derivative medium with 1 mM KNO3 as N source, at 3 weeks after M. loti inoculation. Mature flowers have been obtained from Lotus plants grown in the growth chamber. S=Stems; L = Leaves; R = Roots; N=Nodules; F=Flowers. Data bars represent the mean and standard deviations of data obtained with RNAs extracted from three different sets of plants and 3 RT-qPCR experiments
Fig. 2. Representative spatial profile of the LjNPF2.9 promoter activity in transgenic hairy roots. a-c GUS activity in vascular bundle of mature (a) and young root regions (b and c). d cross section of a stained hairy root. Rp = root primordium; e = endodermis; p = pericycle; ph = phloem; xy = xylem
Fig. 3. a exon/intron organization of the LjNPF2.9 gene. Insertion sites, couple of primers (black and grey; Additional file 8: Table S4) used for expression analyses and relative orientations of the LORE1 retrotransposon element in the 30,086,034, 30,071,286 and 30,007,925 lines are indicated; b expression analysis of the LjNPF2.9 gene. Total RNAs isolated from root tissues of the wild type and ljnpf2.9 mutants obtained from the three different LORE1 lines has been used for RT-PCR analysis (two different ljnpf2.9–1 mutants, a and b are shown)
Fig. 4. Shoot biomass parameters of wild type and ljnpf2.9–1 mutants grown on different KNO3 conditions. a and b shoots length at 2 and 3 weeks after sowing; c and d fresh shoots weight at 2 and 3 weeks after sowing. Plant genotypes and KNO3 concentrations are indicated. Data bars represent the mean and standard errors obtained from 3 independent experiments (10 plants per experiment). Asterisks indicate significant differences with wild type values: *p < 0.02; ** < 0.0007
Fig. 5. Shoots weight/unit length of wild type and ljnpf2.9 mutants grown in the presence of different N sources. Plant genotypes and N sources concentrations are indicated. 0.5 mM ammonium succinate (NH4OOC(CH2)COONH4) is equivalent to 1 mM ammonium. Data bars represent the mean and standard errors obtained from 3 independent experiments (10 plants per experiment). Asterisks indicate significant difference with wild type values: *p < 0.0001; **p < 0.001; ***p < 0.002
Fig. 6. Morphometric measurements of leaves of wild type, ljnpf2.9–1 and ljnpf2.9–3 mutants grown in the presence of 1 mM and 10 mM KNO3. a leaf area (n = 60). Correspondent trifolia from 20 plants for each genotype have been analyzed. Bars represent mean and standard errors; b cell size (equivalent diameter) of leaves (n = 18). Correspondent trifolia from 3 plants for each genotype have been detached and analyzed. sl = superior lamina; il = inferior lamina. Bars represent the common standard error from two factors ANOVA. Different plant genotypes are indicated. Asterisks indicate significant differences (*p < 0.001;** p < 0.0001)
Fig. 7. Functional expression of LjNPF2.9 in Xenopus laevis oocytes in 10 mM external nitrate at pH 5.5 and 6.5. Nitrate accumulation in control oocytes injected with water (white bars), with complementary RNAs expressing LjNPF2.9 (black bars) and AtNPF6.3 (grey bars) (n = 5–8). Values are means ± SE
Fig. 8. a and b nitrate content of shoots and roots of wild type and ljnpf2.9 mutants. Plants are grown on 10 mM KNO3 and analyzed 3 weeks after sowing; c time course of the nitrate content in shoots of wild type and ljnpf2.9–1 mutants. Plants grown for 2 weeks on glutamine 1 mM are transferred on 10 mM KNO3 and analyzed at different time points (0, 1, 2 and 4 days). Data bars represent means and SE from three independent samples (10 plants per sample). Bars corresponding to wild type and mutants plants are indicated
Fig. 9. Symbiotic phenotypes of wild type and ljnpf2.9–1 mutants. a number of nodules per plant. Mature nodules have been scored at 4 weeks post inoculation with M. loti. KNO3 concentrations are indicated; b acetylene reduction activity (ARA) per fresh nodule weight. Plants have been grown in the presence of 1 mM KNO3 and nodules analyzed at 4 weeks post infection. Plant genotypes are indicated. Data bars represent the mean and standard errors obtained from 3 independent experiments (10 plants per experiment)
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