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Proanthocyanidin oxidation of Arabidopsis seeds is altered in mutant of the high-affinity nitrate transporter NRT2.7.

David LC, Dechorgnat J, Berquin P, Routaboul JM, Debeaujon I, Daniel-Vedele F, Ferrario-Méry S - J. Exp. Bot. (2014)

Bottom Line: This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation.Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype.All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.

View Article: PubMed Central - PubMed

Affiliation: Institut Jean-Pierre Bourgin (IJPB), UMR 1318 INRA-AgroParisTech, Centre de Versailles-Grignon, Route de St-Cyr (RD10), F-78026 Versailles cedex, France.

ABSTRACT
NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.

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Nitrate content and gene expression in developing seeds from 9 to 21 d after flowering (DAF). (A, B) Nitrate content of nrt2.7-2 and wild type in excised seeds (Ws) (A) and in siliques emptied from their seeds (silique tissues) (B). (C, D) Expression of TT10, NRT2.7, and CLCa of Ws in excised seeds (A) and silique tissues (B). Each gene expression data was normalized to the level of a synthetic reference gene (SRG) using reference genes EF1a and APC, as described in Materials and methods. Values are mean±standard error of seeds of three individual plants. NA, not analysed.
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Figure 5: Nitrate content and gene expression in developing seeds from 9 to 21 d after flowering (DAF). (A, B) Nitrate content of nrt2.7-2 and wild type in excised seeds (Ws) (A) and in siliques emptied from their seeds (silique tissues) (B). (C, D) Expression of TT10, NRT2.7, and CLCa of Ws in excised seeds (A) and silique tissues (B). Each gene expression data was normalized to the level of a synthetic reference gene (SRG) using reference genes EF1a and APC, as described in Materials and methods. Values are mean±standard error of seeds of three individual plants. NA, not analysed.

Mentions: It has already been described that PA oxidation in the testa starts with the desiccation of developing seeds (Pourcel et al., 2005). In order to better understand the link between NRT2.7 and PA oxidation/accumulation in seeds, the current work investigated more precisely the fluctuation of NO3– content in seeds and in siliques tissues (siliques excluding seeds) during seed development. The NO3– content was the highest in young seeds (9 DAF) and decreased abruptly (12 DAF) to the final low content in mature seeds (Fig. 5A). Conversely NO3– content was the lowest in young siliques tissues (9 DAF) and increased regularly up to the senescing stage (21 DAF) (Fig. 5B). In the nrt2.7-2 mutant, the NO3– contents were slightly lowered in seeds at 12 DAF and in mature seeds compared to those in Ws (Fig. 5A), concomitantly to the maxima of NRT2.7 expression in Ws (Fig. 5C). In contrast, NO3– content was not affected in silique tissues of the nrt2.7-2 mutant (Fig. 5B). Thus, NRT2.7 was likely not the only actor responsible for NO3– accumulation in these tissues. According to Almagro et al. (2008), the impact of the NRT1.6 (AtNPF2.12) mutation was strongly associated with a reduced NO3– content in seeds and an increased seed abortion, but no colour phenotype of the nrt1.6 mutant seeds was reported. In the current study, no significant difference in NRT1.6 (AtNPF2.12) expression was measured in Ws and in nrt2.7-2 (data not shown). NRT1.6 (AtNPF2.12) was expressed in the vascular tissue of the silique and funiculus and was partially responsible for the delivery of NO3– into the seed, but NRT1.6 (AtNPF2.12) was localized at the plasma membrane and, thus, may not be able to compensate the vacuolar nitrate storage in nrt2.7-2. Expression of the vacuolar anionic channel CLCa was detected in silique tissues (Fig. 5D) and, thus, could explain the partial compensation mechanism for the loss of NRT2.7 function in this organ, but no expression of CLCa was measured in excised seeds (Fig. 5C). Further study is required to find out if any other transporter is functional in these organs.


Proanthocyanidin oxidation of Arabidopsis seeds is altered in mutant of the high-affinity nitrate transporter NRT2.7.

David LC, Dechorgnat J, Berquin P, Routaboul JM, Debeaujon I, Daniel-Vedele F, Ferrario-Méry S - J. Exp. Bot. (2014)

Nitrate content and gene expression in developing seeds from 9 to 21 d after flowering (DAF). (A, B) Nitrate content of nrt2.7-2 and wild type in excised seeds (Ws) (A) and in siliques emptied from their seeds (silique tissues) (B). (C, D) Expression of TT10, NRT2.7, and CLCa of Ws in excised seeds (A) and silique tissues (B). Each gene expression data was normalized to the level of a synthetic reference gene (SRG) using reference genes EF1a and APC, as described in Materials and methods. Values are mean±standard error of seeds of three individual plants. NA, not analysed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
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Figure 5: Nitrate content and gene expression in developing seeds from 9 to 21 d after flowering (DAF). (A, B) Nitrate content of nrt2.7-2 and wild type in excised seeds (Ws) (A) and in siliques emptied from their seeds (silique tissues) (B). (C, D) Expression of TT10, NRT2.7, and CLCa of Ws in excised seeds (A) and silique tissues (B). Each gene expression data was normalized to the level of a synthetic reference gene (SRG) using reference genes EF1a and APC, as described in Materials and methods. Values are mean±standard error of seeds of three individual plants. NA, not analysed.
Mentions: It has already been described that PA oxidation in the testa starts with the desiccation of developing seeds (Pourcel et al., 2005). In order to better understand the link between NRT2.7 and PA oxidation/accumulation in seeds, the current work investigated more precisely the fluctuation of NO3– content in seeds and in siliques tissues (siliques excluding seeds) during seed development. The NO3– content was the highest in young seeds (9 DAF) and decreased abruptly (12 DAF) to the final low content in mature seeds (Fig. 5A). Conversely NO3– content was the lowest in young siliques tissues (9 DAF) and increased regularly up to the senescing stage (21 DAF) (Fig. 5B). In the nrt2.7-2 mutant, the NO3– contents were slightly lowered in seeds at 12 DAF and in mature seeds compared to those in Ws (Fig. 5A), concomitantly to the maxima of NRT2.7 expression in Ws (Fig. 5C). In contrast, NO3– content was not affected in silique tissues of the nrt2.7-2 mutant (Fig. 5B). Thus, NRT2.7 was likely not the only actor responsible for NO3– accumulation in these tissues. According to Almagro et al. (2008), the impact of the NRT1.6 (AtNPF2.12) mutation was strongly associated with a reduced NO3– content in seeds and an increased seed abortion, but no colour phenotype of the nrt1.6 mutant seeds was reported. In the current study, no significant difference in NRT1.6 (AtNPF2.12) expression was measured in Ws and in nrt2.7-2 (data not shown). NRT1.6 (AtNPF2.12) was expressed in the vascular tissue of the silique and funiculus and was partially responsible for the delivery of NO3– into the seed, but NRT1.6 (AtNPF2.12) was localized at the plasma membrane and, thus, may not be able to compensate the vacuolar nitrate storage in nrt2.7-2. Expression of the vacuolar anionic channel CLCa was detected in silique tissues (Fig. 5D) and, thus, could explain the partial compensation mechanism for the loss of NRT2.7 function in this organ, but no expression of CLCa was measured in excised seeds (Fig. 5C). Further study is required to find out if any other transporter is functional in these organs.

Bottom Line: This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation.Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype.All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.

View Article: PubMed Central - PubMed

Affiliation: Institut Jean-Pierre Bourgin (IJPB), UMR 1318 INRA-AgroParisTech, Centre de Versailles-Grignon, Route de St-Cyr (RD10), F-78026 Versailles cedex, France.

ABSTRACT
NRT2.7 is a seed-specific high-affinity nitrate transporter controlling nitrate content in Arabidopsis mature seeds. The objective of this work was to analyse further the consequences of the nrt2.7 mutation for the seed metabolism. This work describes a new phenotype for the nrt2.7-2 mutant allele in the Wassilewskija accession, which exhibited a distinctive pale-brown seed coat that is usually associated with a defect in flavonoid oxidation. Indeed, this phenotype resembled those of tt10 mutant seeds defective in the laccase-like enzyme TT10/LAC15, which is involved in the oxidative polymerization of flavonoids such as the proantocyanidins (PAs) (i.e. epicatechin monomers and PA oligomers) and flavonol glycosides. nrt2.7-2 and tt10-2 mutant seeds displayed the same higher accumulation of PAs, but were partially distinct, since flavonol glycoside accumulation was not affected in the nrt2.7-2 seeds. Moreover, measurement of in situ laccase activity excluded a possibility of the nrt2.7-2 mutation affecting the TT10 enzymic activity at the early stage of seed development. Functional complementation of the nrt2.7-2 mutant by overexpression of a full-length NRT2.7 cDNA clearly demonstrated the link between the nrt2.7 mutation and the PA phenotype. However, the PA-related phenotype of nrt2.7-2 seeds was not strictly correlated to the nitrate content of seeds. No correlation was observed when nitrate was lowered in seeds due to limited nitrate nutrition of plants or to lower nitrate storage capacity in leaves of clca mutants deficient in the vacuolar anionic channel CLCa. All together, the results highlight a hitherto-unknown function of NRT2.7 in PA accumulation/oxidation.

Show MeSH