Genome-wide identification and analyses of tomato (Solanum lycopersicum L.) high-affinity nitrate transporter 2 (NRT2) family genes and their responses to drought and salinity

High-affinity nitrate transporter 2 (NRT2) proteins have vital roles in nitrate (NO3-) uptake and translocation in plants. The gene families coding NRT2 proteins have been identified and functionally characterized in many plant species. However, no systematic identification of NRT2 family members have been reported in tomato (Solanum lycopersicum). There is also little known about their expression profiles under environmental stresses. Accordingly, the present study aimed to identify NRT2 gene family in the tomato genome; then, investigate them in detail through bioinformatics, physiological and expression analyses. As a result, four novel NRT2 genes were identified in the tomato genome, all of which contain the same domain belonging to the Major Facilitator Superfamily (PF07690). The co-expression network of SlNRT genes revealed that they were co-expressed with several other genes in many different molecular pathways including transport, photosynthesis, fatty acid metabolism and amino acid catabolism. Programming many crucial physiological and metabolic pathways, various numbers of phosphorylation sites were predicted in the NRT2 proteins.

Using Transcript Levels of Nitrate Transporter 2 as Molecular Indicators to Estimate the Potentials of Nitrate Transport in Symbiodinium, Cladocopium, and Durusdinium of the Fluted Giant Clam, Tridacna squamosa

Giant clams are important ecosystem engineers of coral reefs because they harbor large quantities of phototrophic Symbiodiniaceae dinoflagellates of mainly genera Symbiodinium, Cladocopium, and Durusdinium. The coccoid dinoflagellates donate photosynthate and amino acids to the clam host, which in return needs to supply inorganic carbon and nitrogen to them. The host can conduct light-enhanced absorption of nitrate (NO3–), which can only be metabolized by the symbionts. This study aimed to clone nitrate transporter 2 (NRT2) from the symbionts of the fluted giant clam, Tridacna squamosa. Here, we report three major sequences of NRT2 derived from Symbiodinium (Symb-NRT2), Cladocopium (Clad-NRT2) and Durusdinium (Duru-NRT2). Phenogramic analysis and molecular characterization confirmed that these three sequences were NRT2s derived from dinoflagellates. Immunofluorescence microscopy localized NRT2 at the plasma membrane and cytoplasmic vesicles of the symbiotic dinoflagellates, indicating that it could partake in the uptake and transport of NO3–. Therefore, the transcript levels of Symb-NRT2, Clad-NRT2, and Duru-NRT2 could be used as molecular indicators to estimate the potential of NO3– transport in five organs of 13 T. squamosa individuals. The transcript levels of form II ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcII) of Symbiodinium (Symb-rbcII), Cladocopium (Clad-rbcII) and Durusdinium (Duru-rbcII) were also determined in order to calculate the transcript ratios of Symb-NRT2/Symb-rbcII, Clad-NRT2/Clad-rbcII, and Duru-NRT2/Duru-rbcII. These ratios expressed the potentials of NO3– transport with reference to the phototrophic potentials in a certain genus of coccoid dinoflagellate independent of its quantity. Results obtained indicate that Symbiodinium generally had a higher potential of NO3– transport than Cladocopium and Durusdinium at the genus level. Furthermore, some phylotypes (species) of Symbiodinium, particularly those in the colorful outer mantle, had very high Symb-NRT2/Symb-rbcII ratio (7–13), indicating that they specialized in NO3– uptake and nitrogen metabolism. Overall, our results indicate for the first time that different phylotypes of Symbiodiniaceae dinoflagellates could have dissimilar abilities to absorb and assimilate NO3–, alluding to their functional diversity at the genus and species levels.

Phylogenomic and Microsynteny Analysis Provides Evidence of Genome Arrangements of High-Affinity Nitrate Transporter Gene Families of Plants

Nitrate transporter 2 (NRT2) and NRT3 or nitrate-assimilation-related 2 (NAR2) proteins families form a two-component, high-affinity nitrate transport system, which is essential for the acquisition of nitrate from soils with low N availability. An extensive phylogenomic analysis across land plants for these families has not been performed. In this study, we performed a microsynteny and orthology analysis on the NRT2 and NRT3 genes families across 132 plants (Sensu lato) to decipher their evolutionary history. We identified significant differences in the number of sequences per taxonomic group and different genomic contexts within the NRT2 family that might have contributed to N acquisition by the plants. We hypothesized that the greater losses of NRT2 sequences correlate with specialized ecological adaptations, such as aquatic, epiphytic, and carnivory lifestyles. We also detected expansion on the NRT2 family in specific lineages that could be a source of key innovations for colonizing contrasting niches in N availability. Microsyntenic analysis on NRT3 family showed a deep conservation on land plants, suggesting a high evolutionary constraint to preserve their function. Our study provides novel information that could be used as guide for functional characterization of these gene families across plant lineages.

The MdABI5 transcription factor interacts with the MdNRT1.5/MdNPF7.3 promoter to fine-tune nitrate transport from roots to shoots in apple

Nitrate is a major nitrogen resource for plant growth and development and acts as both a crucial nutrient and a signaling molecule for plants; hence, understanding nitrate signaling is important for crop production. Abscisic acid (ABA) has been demonstrated to be involved in nitrate signaling, but the underlying mechanism is largely unknown in apple. In this study, we found that exogenous ABA inhibited the transport of nitrate from roots to shoots in apple, and the transcription of the nitrate transporter MdNRT1.5/MdNPF7.3 was noticeably reduced at the transcriptional level by ABA, which inhibited the transport of nitrate from roots to shoots. Then, it was found that the ABA-responsive transcription factor MdABI5 bound directly to the ABRE recognition site of the MdNRT1.5 promoter and suppressed its expression. Overexpression of MdABI5 inhibited ABA-mediated transport of nitrate from roots to shoots. Overall, these results demonstrate that MdABI5 regulates the transport of nitrate from roots to shoots partially by mediating the expression of MdNRT1.5, illuminating the molecular mechanism by which ABA regulates nitrate transport in apple.

PDF1.5 Enhances Adaptation to Low Nitrogen Levels and Cadmium Stress

Environmental acclimation ability plays a key role in plant growth, although the mechanism remains unclear. Here, we determined the involvement of Arabidopsis thaliana PLANT DEFENSIN 1 gene AtPDF1.5 in the adaptation to low nitrogen (LN) levels and cadmium (Cd) stress. Histochemical analysis revealed that AtPDF1.5 was mainly expressed in the nodes and carpopodium and was significantly induced in plants exposed to LN conditions and Cd stress. Subcellular localization analysis revealed that AtPDF1.5 was cell wall- and cytoplasm-localized. AtPDF1.5 overexpression significantly enhanced adaptation to LN and Cd stress and enhanced the distribution of metallic elements. The functional disruption of AtPDF1.5 reduced adaptations to LN and Cd stress and impaired metal distribution. Under LN conditions, the nitrate transporter AtNRT1.5 expression was upregulated. Nitrate transporter AtNRT1.8 expression was downregulated when AtPDF1.5 was overexpressed, resulting in enhanced transport of NO3− to shoots. In response to Cd treatment, AtPDF1.5 regulated the expression of metal transporter genes AtHMP07, AtNRAMP4, AtNRAMP1, and AtHIPP3, resulting in higher Cd accumulation in the shoots. We conclude that AtPDF1.5 is involved in the processing or transmission of signal substances and plays an important role in the remediation of Cd pollution and LN adaptation.