TaNRAMP3 is essential for manganese transport in Triticum aestivum

Abstract

Manganese (Mn) is an essential trace element for almost all living organisms. In plants, Mn deficiency, which is occurs in calcareous soils or alkaline soils, severely limiting crop yields. However, the potential mechanism of Mn transport in Triticum aestivum is still obscure. Here, we found that TaNRAMP3, a member of the naturally resistant macrophage protein (NRAMP) family in Triticum aestivum, is located in the plasma membrane of protoplasts and functions as an influx transporter for Mn in yeast (Δsmf1). The expression of TaNRAMP3 was induced under Mn-deficiency conditions. Furthermore, TaNRAMP3-RNAi plants exhibited a sensitive phenotype, while transgenic plants overexpressing TaNRAMP3 showed a tolerant phenotype. In addition, TaNRAMP3 rescued the sensitive phenotype of Arabidopsis nramp1 mutant under Mn deficiency condition. In summary, our study reveals the key role of TaNRAMP3 in Mn transport in Triticum aestivum, allowing it to adapt to Mn-deficiency stress. These findings provide new insights for the cultivation of Mn-deficiency tolerant wheat varieties.

Keywords: Manganese; TaNRAMP3; Transporter; Triticum aestivum.

Fig. 1

Subcellular localization of TaNRAMP3 in Triticum aestivum. A Plasma membrane localization of TaNRAMP3 in Triticum aestivum mesophyll protoplasts. TaNRAMP3 is fused to GFP. The PM marker is TaSTP6-mCherry. Scale bars, 5 μm. B Fluorescence analysis of the localization of the interaction between TaNRAMP3-GFP and TaSTP6-mCherry in Triticum aestivum mesophyll protoplasts. The fluorescence intensity (mCherry and GFP signals) of the section lines was scanned using Fiji/ImageJ software. The location of the section line is marked red arrow in (A)

Fig. 2

Functional analysis of TaNRAMP3 on ion transport in yeast. A Potential function analysis of TaNRAMP3 on Mn transport. Growth of yeast strains on normal medium (SD-U, lacking Ura) and treatment medium (SD-U + 100 mM EGTA + 5 mM Ca²⁺) for 3–5 d. Four 10-fold series of diluents were established under sterile conditions. B Growth curves of yeast cells were plotted against OD600 values in normal SD-U nutrient solution. C Growth curves of yeast cells were plotted against OD600 values in SD-U nutrient solution containing 2 mM EGTA. D Potential function analysis of TaNRAMP3 on Mn transport. Growth of yeast strains on normal medium (SD-U, lacking Ura) and treatment medium (SD-U + 80 μM BPDS) for 3–5 d. Four 10-fold series of diluents were established under sterile conditions. E Growth curves of yeast cells were plotted against OD600 values in normal SD-U nutrient solution. F Growth curves of yeast cells were plotted against OD600 values in SD-U nutrient solution containing 20 μM BPDS. G Potential function analysis of TaNRAMP3 on Mn transport. Growth of yeast strains on normal medium (SD-U, lacking Ura) and treatment medium (SD-U + 5 mM EGTA) for 3–5 d. Four 10-fold series of diluents were established under sterile conditions. H Growth curves of yeast cells were plotted against OD600 values in normal SD-U nutrient solution. I Growth curves of yeast cells were plotted against OD600 values in SD-U nutrient solution containing 1 mM EGTA

Fig. 3

Phenotypic analysis of overexpressed plants. A Mn deficiency phenotypes of TaNRAMP3-OE plants. Scale bars, 5 cm. B Statistical analysis of fresh weight of plants shown in (A). C Chlorophyll a content analysis of TaNRAMP3-OE and WT plants shown in (A). The results are presented as the mean ± SD (n = 4 biological replicates, 4 shoots). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05) for genotypes within a given growth condition (+ Mn or -Mn). D Chlorophyll b content analysis of TaNRAMP3-OE and WT plants shown in (A). The results are presented as the mean ± SD (n = 4 biological replicates, 4 shoots). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05) for genotypes within a given growth condition (+ Mn or -Mn). E Mn content analysis of the TaNRAMP3-OE and WT plants. The results are presented as the mean ± SD (n = 3 biological replicates, 9 roots or 9 shoots). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05) for genotypes within a given growth condition (+ Mn or -Mn)

Fig. 4

Phenotypic analysis of TaNRAMP3-RNAi plants. A Mn deficiency phenotypes of TaNRAMP3-RNAi plants. Scale bars, 5 cm. B Statistical analysis of fresh weight of plants shown in (A). C Chlorophyll a content analysis of TaNRAMP3-RNAi and WT plants shown in (A). The results are presented as the mean ± SD (n = 4 biological replicates, 4 shoots). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05) for genotypes within a given growth condition (+ Mn or -Mn). D Chlorophyll b content analysis of TaNRAMP3-OE and WT plants shown in (A). The results are presented as the mean ± SD (n = 4 biological replicates, 4 shoots). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05) for genotypes within a given growth condition (+ Mn or -Mn). E Mn content analysis of the TaNRAMP3-OE and WT plants. The results are presented as the mean ± SD (n = 3 biological replicates, 9 roots or 9 shoots). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05) for genotypes within a given growth condition (+ Mn or -Mn)

Fig. 5

Potential functional analysis of AtNRAMP1 and TaNRAMP3 in Arabidopsis. A Mn deficiency phenotypes of WT, nramp1 and nramp1/TaNRAMP3 transgenic plants. Scale bars, 1 cm. B Statistical analysis of root lengths of plants shown in (A). The data are presented as the mean ± SD (n = 15 seedlings for each genotype). C Statistical analysis of fresh weight of plants shown in (A). The data are presented as the mean ± SD (n = 15 seedlings for each genotype). D Mn deficiency phenotypes of WT, nramp1 and WT/TaNRAMP3 transgenic plants. Scale bars, 1 cm. E Statistical analysis of root lengths of plants shown in (A). The data are presented as the mean ± SD (n = 15 seedlings for each genotype). F Statistical analysis of fresh weight of plants shown in (A). The data are presented as the mean ± SD (n = 15 seedlings for each genotype)