Cloning and Characterization of OsLCT2
Rice OsLCT1 is an important Cd transporter (Uraguchi et al. 2011, 2014). Unexpectedly, no OsLCT1 fragments could be obtained by PCR amplification from the DNA of indica rice cultivars, Huazhan, 93-11 and Shuhui 498. Hence, BLAST searches were conducted using the amino acid sequences of OsLCT1 against the genome of the rice cultivar Shuhui498 in the National Center for Biotechnology Information (NCBI) database. The OsLCT1 sequence could not be aligned, confirming the absence of OsLCT1 in the genome of Shuhui498. However, we found a putative rice LCT protein, designated as OsLCT2. Because the full-length cDNA sequence of OsLCT2 was not available in the Rice Annotation Project Database (http://rapdb.dna.affrc.go.jp/), we performed 5′- and 3′- rapid amplification of cDNA ends (RACE) to clone the full-length cDNA of OsLCT2 (GenBank accession number: MW757982) from the indica rice cultivar Huazhan.
OsLCT2 contains three exons and two introns (Fig. 1a), and encodes a peptide of 478 amino acids. It is located at position 23,774,023 to 23,776,721 on chromosome 6 in the Shuhui498 genome. Similar to TaLCT1 and OsLCT1, OsLCT2 has a hydrophilic amino N terminus containing sequences enriched in Pro, Ser, Thr, and Glu, collectively known as the PEST sequence (Fig. 1b) (Kennedy and Rinne 1997). Both the TMPRED and CCTOP programs predicted that OsLCT2 is a membrane protein with eleven transmembrane domains (Fig. 1b). Phylogenetic analysis showed that LCT-like proteins were only found in grass plants (Fig. 1c). Among those LCT members in grass plants, the closest homolog of OsLCT2 was TaLCT1, which also belonged to the same subgroup as OsLCT2 (Fig. 1c). Amino acid sequence alignment revealed that OsLCT2 shared 55% and 33% sequence identity with TaLCT1 and OsLCT1, respectively. To explore the evolutionary relationship between OsLCT2 and OsLCT1, phylogenetic analysis was conducted using their predicted amino acid sequences and LCT-like proteins in eight wild Oryza species. OsLCT2 and its orthologs in Oryza rufipogon were in the same clade with 98.5% sequence identity, whereas the OsLCT1 showed high similarity to the LCT-like proteins in Oryza barthii, Oryza glaberrima, and Oryza nivara, with 98.6%, 98.6%, and 95.7% sequence identity, respectively (Additional file 1: Fig. S1a). These results revealed that orthologs of both OsLCT2 and OsLCT1 existed in wild rice species, and the considerable sequence divergence between OsLCT2 and OsLCT1 derived from the divergence of their ancestor gene pools. This finding implied that they diverged before the diversification of Oryza genus.
We further analyzed natural variations of OsLCT2 coding sequence (CDS) using 3,620 rice accessions of the MBKbase-rice database (Peng et al. 2020). Twelve single-nucleotide polymorphisms (SNPs) were detected in the OsLCT2 CDS region (Additional file 1: Fig. S1b). Based on these variations, its CDS region was classified into nine haplotypes (rare haplotypes of < 10 accessions were not shown). Haplotype 1, which was harbored in Huazhan and Shuhui 498, was also prevalent in the four main rice subpopulations, accounting for 68.3% of indica, 43.3% of temperate japonica, 72.3% of tropical japonica, and 39.6% of aus (Additional file 1: Fig. S1b, c). Notably, there were different proportions of OsLCT2 deletions in these rice subpopulations. OsLCT2 was absent in about 54% of temperate japonica and aus, but only in a small fraction of indica (4.3%) and tropical japonica (14.3%) (Additional file 1: Fig. S1b, c).
Expression Pattern of OsLCT2
The expression profiles of OsLCT2 in rice seedlings were investigated by quantitative real-time PCR (qRT-PCR). The expression level of OsLCT2 in roots was relatively high, followed by leaves, with the lowest expression level in the basal region (2 cm above the roots) (Fig. 2a). To investigate the effects of metal deficiency and excess on the expression of OsLCT2, rice plants were grown in nutrient solutions either free of Fe, Mn, or Zn or containing high concentrations of Fe, Mn, or Zn for 7 d. Expression of OsLCT2 was unaffected by the deficiency of those metals, while it was induced by excess Fe and Zn (Fig. 2b). To determine the responses of OsLCT2 to Cd stress, rice seedlings were treated with 0, 0.5, 2.5 or 25 μM Cd for 24 h. The OsLCT2 expression in roots was induced by Cd in a dose-dependent manner. Its transcripts increased by about fourfold in response to 25 μM Cd, but slightly increased in response to 2.5 μM Cd concentrations when compared with that in the absence of Cd (Fig. 2c).
To analyze the tissue specificity of OsLCT2 expression, we determined OsLCT2 promoter activity by assessing β-glucuronidase (GUS) activity histochemically in transgenic rice plants expressing the GUS gene driven by the OsLCT2 promoter. The GUS staining was mainly observed in the elongation and maturation zones of the root but not in the meristematic zone and root cap (Fig. 2d I, II). In the cross sections and vertical sections of the elongation and maturation zone of the primary root, GUS activity appeared in all the tissues, including epidermis, cortex and stele, with the strongest signal in pericycle and stele cells adjacent to the xylem (Fig. 2d IV–VII). As the primary root matured, the GUS staining faded away, except for a pronounced staining in the lateral roots (Fig. 2d I, III). The observations in the cross sections and vertical sections of the maturation zone where the lateral roots began to form showed that GUS signal occurred mainly in the stele of the primary and lateral roots, especially the undifferentiated vascular tissue of the lateral roots (Fig. 2d VI–VII).
Subcellular Localization of OsLCT2
To detect the subcellular localization of the OsLCT2 protein, OsLCT2 was separately fused to the N-terminus or C-terminus of an enhanced GFP (eGFP) via a 16 amino-acid linker. OsLCT2-eGFP or eGFP-OsLCT2 driven by the constitutive cauliflower mosaic virus 35S promoter were cloned into a transient expression vector and introduced into rice leaf protoplasts. Both OsLCT2-eGFP and eGFP-OsLCT2 fusion proteins were observed at the ER, as evidenced by their fluorescence signals overlapping with the ER retention signal His-Asp-Glu-Leu (HDEL) fused with the red fluorescent protein mCherry (Fig. 3). In contrast, the control vector (containing eGFP alone driven by the 35S promoter) produced green fluorescence in the cytosol and nucleus (Fig. 3).
Overexpression of OsLCT2 Reduces Cd Concentration in Rice Grains
To investigate the physiological role of OsLCT2 in rice, we generated two independent overexpression lines driven by a maize ubiquitin promoter and two independent knockout lines using CRISPR/Cas9 technology. The two overexpression lines greatly enhanced expression of OsLCT2, compared with that of wild-type (WT) plants, according to the qRT-PCR analysis (Fig. 4a). Sequencing confirmed that each knockout line had insertions that caused a frameshift of the coding sequence (Fig. 4b).
When plants were grown in a field contaminated with 0.65 mg/kg Cd, overexpressing OsLCT2 tended to reduce grain yield compared with that of the WT, although the difference was only statistically significant in one overexpression line (Fig. 4c, e; Additional file 1: Fig. S2). In contrast, no apparent changes of morphological phenotype or grain yield were found between the knockout lines and the WT (Fig. 4d, e). We then measured Cd, Mn, Fe, Zn and Cu accumulation in these plants at the maturity stage to evaluate whether OsLCT2 affected metal accumulation in plants grown in field conditions. The Cd concentrations in the brown rice and straw of the overexpression lines were significantly lower by 24.1–27.5% and 30.7–37.5%, respectively, compared with those of the WT (Fig. 4f, k). These results suggest that OsLCT2 overexpression may not only affect Cd in the grain but also in all above-ground tissues. However, the Mn, Fe, Zn and Cu concentrations in brown rice and straw were similar between the overexpression lines and WT (Fig. 4g–j, l–o). In comparison, all of the above metal concentrations in brown rice and straw did not differ significantly between the knockout lines and the WT (Fig. 4f–o).
Overexpression of OsLCT2 Weakens Root-to-Shoot Translocation of Cd, Mn, Fe and Zn
To understand the cause of OsLCT2 overexpression reducing Cd concentrations in rice straw and grains, two-week-old seedlings of hydroponically-grown OsLCT2 overexpression lines and WT plants were transferred into nutrient solutions containing 0, 0.5 or 1 μM Cd and kept in solutions for 14 days. Growth responses and divalent metal concentrations in shoots and roots of the overexpression lines and WT were investigated. OsLCT2 overexpression lines and WT plants showed comparable growth within each of the 0 and 0.5 μM Cd treatments, while shoots of overexpression lines were 7–8% longer and 13–17% heavier (dry weight) in the 1 μM Cd treatment (Fig. 5a–d). The results suggest that overexpressing OsLCT2 does not affect seedling growth and enhances rice tolerance to excess Cd under hydroponic culture with sufficient mineral nutrition.
Analysis of metal concentrations showed that there were no differences in Mg, Ca and Cu concentrations in both the shoots and roots between the overexpression lines and WT plants (Fig. 5e–h), while the Mn, Fe, Zn concentrations in shoots were significantly lower in the overexpression lines than in the WT with and without Cd stress (Fig. 5f; Additional file 1: Fig. S3a–c). The Mn and Fe concentrations in roots were similar between the WT plants and overexpressing lines, whereas the Zn concentration in roots was significantly higher in the overexpression lines than in the WT plants with or without Cd treatment (Fig. 5h; Additional file 1: Fig. S3d, e). Under hydroponic conditions without Cd, growth of the overexpression lines was similar to that of the WT plants (Fig. 5a), suggesting that the reduced concentrations of Mn, Fe, and Zn in shoots were sufficient for the normal growth and development of overexpression lines.
In the presence of 0.5 and 1 μM Cd, OsLCT2 overexpression lines exhibited 21.8–26.9% and 26.7–28.1% lower Cd concentrations in shoots, respectively, but similar Cd concentrations in roots compared to those of the WT (Fig. 5i, j). In the presence of 1 μM Cd, overexpression lines showed better Cd tolerance than the WT, which might be attributed to the lower Cd concentrations in shoots that reduced the inhibitory effect of Cd on growth of plants (Fig. 5a). Besides, the overexpression lines showed lower Cd, Mn, Fe and Zn concentrations in the xylem sap than that of the WT (Fig. 5k). As a result, its overexpression significantly decreased the percentages of Cd, Mn, Fe and Zn translocated from roots to shoots (Fig. 5l). Among them, the proportion of Mn transported to the shoots was slightly lower, while the proportions of Cd, Zn and Fe transported to the shoots were moderately lower than those of the WT (Fig. 5l). These results indicate that OsLCT2 has poor ability to discriminate among the divalent metal cations Cd, Mn, Fe and Zn. Its overexpression weakens the translocation of these four metals from roots to shoots by limiting their amounts loaded into the xylem.
Effect of OsLCT2 Overexpression on Expression of OsZIP Genes
Because the ZIP family displays transport activity of various cations, including Zn, Fe, Mn and Cd (Lee and An 2009; Ajeesh Krishna et al. 2020), we examined expression of ten genes belonging to the OsZIP family in roots of OsLCT2 overexpression lines and the WT in the presence or absence of 0.5 μM Cd for 24 h. Among the genes, the expression levels of OsIRT1, OsIRT2, OsZIP5, OsZIP6 and OsZIP9 were upregulated by Cd in both the WT and OsLCT2 overexpression lines (Fig. 6). The expression levels of most OsZIP genes did not differ between the OsLCT2 overexpression and WT plants, with the exception of OsZIP9 expression, which was significantly higher in the overexpression lines than in the WT, irrespective of Cd stress (Fig. 6). OsZIP9 was recently reported to play an important role in the uptake of Zn in rice (Huang et al. 2020; Tan et al. 2020; Yang et al. 2020), so the increased concentrations of Zn in roots of overexpression lines may have been caused by the up-regulation of OsZIP9 expression.