Table 1.
Details of the metal tolerance protein (MTP) genes identified in rice genome.
Fig 1.
Phylogenetic relationship, exon-intron structure and identification and distribution of conserved motifs of rice metal tolerance proteins (MTPs).
(a) The un-rooted phylogenetic tree was constructed via alignment of full-length amino acid sequences from rice using MEGA 7.0 software by using Maximum-likelihood method. Lengths of the exons and introns of each MTP gene are displayed proportionally. Yellow rectangles and thin lines indicate exons and introns, respectively. The un-translated regions (UTRs) are indicated by blue rectangles. (b) All motifs were identified by Multiple EM for Motif Elicitation (MEME) tool using the complete amino acid sequences of OsMTP proteins. Different colored boxes indicate different motifs. Only highlighted motifs are searched by MEME software and the annotation of each motif is listed at the bottom panel. The non-highlighted motifs are searched by other motif finding software.
Table 2.
Single nucleotide polymorphism (SNP) for metal tolerance protein genes in three thousand rice genotypes.
Table 3.
Genetic diversity analysis of MTP genes in three thousand rice genotypes.
Fig 2.
Predicted 3D models of rice MTP proteins.
Models were generated by using Phyre2 protein-modeling server at intensive mode. The protein 3D structures were further refined using ModRefiner.
Table 4.
Presence of MTP genes in various rice QTLs/meta-QTLs associated with metal tolerance/accumulation.
Fig 3.
Expression profiling of rice metal tolerance protein (MTP) genes in root and shoot tissues of 1-week, 2-week, 3-week old seedlings.
(a) Q-PCR analysis of MTP transcripts. Transcript levels of the MTP genes were normalized with transcript levels of internal reference gene OsUBQ5. Then the resulting expression level of MTP genes was compared to normalized expression level of constitutively expressed OseEF-1a gene. The resulting levels of expression of MTPs are compared among themselves as well as between roots and shoots. (b) Expression values in FPKM (fragments per kilobases of gene per million mapped reads) obtained from Rice Expression Database (http://expression.ic4r.org/).
Fig 4.
Expression profile of rice MTP genes during reproductive growth.
(a) Q-PCR analysis showing normalized expression of MTP genes in developing seeds at different DAF (Days After Fertilization) stages. Fold change between different time points is calculated against 7 DAF stage. Asterisks indicate T-test results where * = p<0.05, ** = p<0.01, *** = p<0.001. (b) Hierarchical clustering of Q-PCR results showing the expression profile of MTP genes in developing seeds. Relative fold change of MTP expression (normalized against OsUBQ5) was calculated against OseEF-1a expression (normalized against OsUBQ5) gene. The resuting fold change values were then used to make heatmap. The heatmap representing hierarchical clustering of log2 transformed expression values of MTP genes at different developmental stages (indicated at the top of each lane) was generated using MeV software package (http://mev.tm4.org/). The color bar below represents relative expression value from green color representing the lowest expression levels, black represents medium expression levels, and red signifies the highest expression level. (c, d) Expression values of rice MTP genes at various stages of reproductive growth shown as FPKM (fragments per kilobases of gene per million mapped reads) values obtained from Rice Expression Database (http://expression.ic4r.org/). (d) Expression in aleurone layer and effect of GA (Gibberellic Acid) and ABA (Abscisic Acid) on aleuronic expression.
Fig 5.
Q-PCR analysis of gene expression changes of rice MTP genes in response to various heavy metal stress conditions.
Q-PCR analysis of MTP genes in roots (a), and shoots (b), in response to 24-hour exposure to Co, Cd, and Ni. The fold change is calculated against untreated samples. Asterisks indicate Student’s T-test results, where * = p<0.05, ** = p<0.01, *** = p<0.001.
Fig 6.
Gene expression changes of rice MTP genes in response to starvation conditions of Zn and Fe.
Q-PCR analysis of MTP genes in roots (a, c) and shoots (b, d) in response to 1 week (a, b) or 2 week (c, d) of Zn or Fe deficiency conditions. The fold change is calculated against untreated samples. Asterisks indicate T-test results where * = p<0.05, ** = p<0.01, *** = p<0.001.
Table 5.
Functional effect prediction for the amino acid changes located in the candidate OsMTP8 gene.
Neutral or deleterious effect was predicted based on the score estimated using PROVEAN tool (http://provean.jcvi.org/about.php).