Expression pattern of OsNRAMP2
Quantitative reverse transcription PCR (Q-RT-PCR) was used to determine the expression levels of OsNRAMP2 in various tissues of rice plants (cv. Nipponbare) grown in a paddy field at different growth stages. At the seedling, tillering, booting and flowering stages, OsNRAMP2 was mainly expressed in the roots, leaf sheaths and leaf blades (Fig. 1A). The expression levels of OsNRAMP2 in the leaf sheaths and leaf blades were higher than those in the rootsand stems at all growth stages. The expression level of OsNRAMP2 in the leaf sheaths and leaf blades generally increased with growth stage with a peak at the flowering stage (Fig. 1A).
The responses of OsNRAMP2 transcript level to withdrawal of micronutrients Fe, Zn and Cu or the addition of Cd were investigated in a hydroponic experiment. Withdrawal of Fe for 7 days slightly induced the expression of OsNRAMP2 in roots, whereas other treatments had little effect (Fig. 2B). The response to Fe deficiency was further investigated in a time-course experiment. In both roots and shoots, OsNRAMP2 expression was induced by Fe deficiency from day 2 onward, with a 1.75- and 2-fold increase, respectively, on day 5 of Fe withdrawal compared with the +Fe treatment on day 0 (Fig. 2C).
Tissue specificity and subcellular localization of OsNRAMP2
To investigate the tissue and cell specificity of OsNRAMP2 expression, we generated transgenic rice (cv. ZH11) expressing the GUS reporter gene under the control of the native promoter of OsNRAMP2. Histochemical staining of the GUS activity in the pOsNRAMP2::GUS transgenic lines showed that OsNRAMP2 was expressed mainly in the radicle and embryo of germinated seeds, as well as roots, leaf sheaths and leaf blades of seedings (Fig. 2A, B, C, H). Consistent with the Q-RT-PCR data, withdrawal of Fe from the nutrient solution for 7 days slightly increased the GUS activity in the roots (Fig. 2D, E, F, G). The GUS activity was localized in all root cells, as well as in the leaf mesophyll cells and the parenchyma cells surrounding the vascular bundles in the leaf sheaths (Fig. 2F, G, I).
To determine the subcellular localization of OsNRAMP2, OsNRAMP2-eGFP or eGFP-OsNRAMP2 was transiently expressed in rice protoplasts driven by the CaMV35S promoter. MADS3-mCherry fusion protein was used as a nucleus marker (Gao et al. 2014). In the control expressing eGFP alone, the GFP signal was detected in the cytoplasm, whereas the GFP signal in the protoplasts expressing OsNRAMP2-eGFP or eGFP-OsNRAMP2 was observed at the tonoplast with the nucleus marked by MADS3-mCherry being outside of the GFP signal (Fig. 3). Fusion of GFP to the N- or C-terminal of OsNRAMP2 revealed the same subcellular localization at the tonoplast.
Knockout of OsNRAMP2 inhibited germination and suppressed remobilization of vacuolar Fe during seed germination
Knockout of OsNRAMP2 delayed seed germination (Fig. 4A, B). After 3 days of soaking at 37 oC, two osnramp2 mutants had a germination rate of 21.2 – 27.2%, compared with 86.4% in wild type (Fig. 4C). After seedlings were grown in tap water for 8 days, shoot height of osnramp2 mutants was 39.3% shorter than that of wild type (Fig. 4D). Moreover, osnramp2 mutants displayed leaf chlorotic symptoms typical of Fe deficiency (Fig. 4D). The concentrations of Fe in the shoots and roots of the osnramp2 mutants were significantly (P < 0.05) lower, by 41.2 - 46.9% and 41.9 - 47.8%, respectively, than wild type (Fig. 4E). The lower Fe concentrations in the mutant seedlings were not attributed to lower Fe concentrations in the seeds, because there was no significant difference in seed Fe concentration between mutants and wild type (Fig. 4F).
To investigate whether knockout of OsNRAMP2 affects Fe remobilization during germination, we used electron microscopy coupled to energy dispersive X-ray (EDX) spectra to image the distribution of Fe in the scutellum cells of germinated seeds. In both wild type and osnramp2, Fe was localized to the electron dense globoids inside the protein bodies (i.e. protein storage vacuoles, Fig. 4G, H) in scutellum cells, which was confirmed by EDX analysis of individual globoids (Fig. 4L and M). This pattern of Fe localization is similar to that reported for germinated seeds of Arabidopsis thaliana (Lanquar et al. 2005). Compared with wild type, osnramp2 mutant showed more Fe-containing globoids inside vacuoles (Fig. 4I and K), suggesting that remobilization of Fe from the protein storage vacuoles during germination is restricted in the mutant.
osnramp2 mutants were more sensitive to Fe deficiency at the seedling stage
Seedlings (14-day-old) of two osnramp2 mutants and wild type were grown in nutrient solution containing 0, 0.2 or 20 μM Fe for 21 days. Under low Fe (0.2 μM) or no Fe conditions, osnramp2 mutants grew more poorly than wild type, with significantly shorter root length and shoot height (Figs. 5A, B). Total root length, root surface area and the number of root tips (including both primary and lateral roots) of the mutants were all significantly (P < 0.05) smaller than those of wild type (Figs. 5B, Supplemental S3A-S3E). The youngest fully developed leaves of the mutant lines were chlorotic (Fig. 5A), and the chlorophyll content (SPAD value) was significantly lower than wild type (Fig. 5C). Meanwhile, knockout of OsNRAMP2 significantly decreased plant total biomass (Fig. 5D). Surprisingly, osnramp2 mutants contained significantly higher concentrations of Fe in roots and shoots than wild type under low Fe or no Fe treatments (Fig. 5E, F). Supply of 20 μM Fe alleviated the chlorotic symptoms of osnramp2 mutants and improved their growth, although leaf chlorophyll content and plant biomass were still smaller than wild type (Fig. 5A, C, D). In addition, there was no significant difference in either shoot or root Fe concentration between mutants and wild type in the 20 μM Fe treatment (Fig. 5E, F).
Knockout of OsNRAMP2 increased the expression of Fe transport related genes
Higher concentrations of Fe in the roots and shoots of osnramp2 mutants could be an indirect result from altered expression of genes involved in Fe uptake and transport. To test this hypothesis, we quantified the expression of OsIRT1, OsIRT2, OsYSL15, OsTOM1, OsNAS1 and OsNAAT1 in the roots of mutants and wild type grown under different Fe (0, 0.2 and 20 μM) supply conditions for 21 days. As expected, low Fe or no Fe conditions increased the expression of OsIRT1, OsIRT2, OsYSL15, OsTOM1, OsNAS1 and OsNAAT1 markedly (Fig. 6A-F). Moreover, osnramp2 mutants showed significantly higher expression of OsIRT1 and OsYSL15 than wild type under all Fe supply conditions (Fig. 6A-C). In addition, knockout of OsNRAMP2 significantly increased the expression of OsTOM1, OsNAS1, and OsNAAT1 in either normal or the low Fe conditions (Fig. 6D-F).
Knockout of OsNRAMP2 decreased Cd translocation to and accumulation in rice grain
To test whether OsNRAMP2 is involved in Cd distribution, seedlings (21-day-old) of two osnramp2 mutants and wild type were grown in nutrient solution containing 0.5 or 1.0 μM Cd for 10 days. There were no significant differences in the growth phenotypes including root length and shoot height between mutants and wild type (Supplemental Fig. 45A-C). Knockout of OsNRAMP2 had no significant effect on the concentrations of Cd in the roots and shoots at 0.5 μM Cd (Fig. 7A, B). At 1.0 μM Cd, the concentrations of Cd in the roots and shoots of two osnramp2 lines were significantly (P < 0.05) lower than those of wild type by 20.4 - 22.7% and 11.6 - 13.9%, respectively (Fig. 7A, B). After exposure to 1.0 μM Cd for 12 h, xylem sap collected from two osnramp2 mutants also contained significantly lower concentrations of Cd than that from wild type (Fig. 7C).
To examine the effect of knockout of OsNRAMP2 on Cd accumulation in rice grain, three osnramp2 mutant lines and wild type were grown in a Cd-contaminated paddy soil in a pot experiment. At maturity, there was no significant difference in plant height between osnramp2 mutants and wild type (Fig. 8A). However, knockout of OsNRAMP2 decreased straw biomass and grain yield by 47.3 - 59.7% and 31.5 -47.0%, respectively, compared with wild type (Fig. 8B, C). Grain Cd concentration in wild type was well over the Chinese permissible limit of 0.2 mg kg-1. Knockout of OsNRAMP2 significantly decreased grain Cd concentration by 21.0 - 23.9% compared with wild type (Fig. 8D). In contrast, Cd concentrations in the flag leaves, leaf sheaths and straws of osnramp2 mutants were significantly higher than those of wild type (Fig. 8E, F, G). In contrast, knockout of OsNRAMP2 did not significantly affect the concentrations of Fe in the grains, flag leaves, leaf sheaths or straws, although there was a tendency for higher concentrations in the flag leaves, leaf sheaths and straws of mutants (Fig. 8H-L).