Molecular characterization of mineral stress responsive rice genes and identification of their putative orthologs
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Date
2021-08
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Abstract
Plants majorly experience low phosphorous (P) and high iron (Fe) levels in acidic soils (pH≤5), which leads to reduction in crop productivity. These stresses lead to various changes at physiological and molecular level and induce various stress responsive genes. The aim of this study was to analyze the expression of already reported mineral stress (low P) responsive rice genes such as PHR3, PHO1.2 across diverse and contrasting genotypes such as LR11, LR23, LR26, KMR3, BAM350 and BAM811. Expression of genes like AAO2, PRI reported in high Fe stress response were also analysed in these genotypes using qPCR. The stress conditions such as low Pi (Pi = 0.015 mM/L) and high Fe (Fe2+ = 2.56 mg/L) was given through hydroponic sand culture method along with control (Pi = 0.35 mM; Fe2+ = 0.02 mM) and differential gene expression (analysed by qPCR) after 24 hrs, 48 hrs and 7 days of treatment was evaluated in shoots. The physical parameters such as shoot length, root length, fresh shoot and root weight, dry shoot and root weight were also measured. The selected low P tolerant genotypes performed well under both low P and high Fe conditions. In response to low P conditions, all the tolerant genotypes had lower malondialdehyde (MDA) levels while susceptible genotypes had higher levels. In response to high Fe conditions, tolerant LR23 showed very high levels of MDA. Relative downregulation in expression of PHR3 in response to 24 hrs of low Pi treatment was observed for all the genotypes except BAM811 and LR11. However, in response to 48 hrs of low P stress, the low P tolerant genotypes LR23 and LR11 maintain the expression of PHR3. LR26 show a slight decrease and KMR3 shows a significant increase. The two low P susceptible genotypes BAM350 and BAM811 show a drastic reduction in PHR3 expression levels. PHO1.2 (responsible for transport of P from roots to shoots) expression in response to 24 hr low P treatment in low P tolerant genotypes (LR23 and LR26) was significantly upregulated whereas in LR11 the expression levels were significantly higher and maintained under both abundant and low P conditions. The expression levels were significantly downregulated after 48 hrs of low Pi treatment. KMR3 showed reverse trend for PHO1.2 in response to 24 and 48 hrs of low Pi stress. The expression levels of PHO1.2 in low P conditions were significantly low as compared to control in susceptible genotype BAM811. Genotype BAM350 showed high levels of PHO1.2 only in response to 48 hrs of low P treatment. Our data suggests that the expression of key genes involved in low P tolerance response is either delayed or downregulated. OsPHR3 and PHO1.2 expression has not been checked under high Fe conditions, till date. Our data suggests that PHR3 and PHO1.2 are highly induced in shoots after 48 and 24 hrs of high Fe stress, respectively. The expression levels of PRI (responsible for high accumulation of Fe in plants) in response to 24 hrs of high Fe treatment was significantly upregulated in all the genotypes with expression levels many folds more in LR26 and LR11. After 48 hrs of high Fe treatment, levels of PRI were downregulated in low P tolerant genotypes LR26, LR11 and KMR3. However, LR23 showed high levels of PRI expression. Susceptible genotype BAM350 the levels of PRI were many fold higher in high Fe conditions. Our experiments suggest differential expression of PRI in low P conditions as well. Under high Fe treatment for 7 days, oxidative stress as measured by levels of AAO2 was observed in BAM811. Genotypes LR11 and KMR3 also showed higher levels of AAO2 under Fe stress. The expression levels of AAO2 were higher in LR23, LR11 and KMR3 after 7 days of low P treatment. In low P tolerant genotype LR26, AAO2 downregulation was observed. Low P susceptible genotype BAM350 also showed significant downregulation whereas no change in AAO2 levels was observed for BAM811. In order to understand the role of rice genes involved in mineral stress in other species, orthologs of selected genes were identified in Brassica and linseed using bioinformatics tools. Orthologs of PHR3,
AAO2, PRI in both Brassica and linseed and ortholog of PHO1.2 only in Brassica could be identified. The functional domains such as MYB, Myc, multicopper oxidase, SPX-EXS were all conserved across rice, Brassica and Linum and proteins from these three species could be aligned using ClustalW. The phylogenetic tree generated showed 3 distinct groups where Brassica species (B. napus, B. oleracea, B. rapa) formed one closely related group, and Linum and rice proteins were placed as two separate groups. An attempt to amplify the putative orthologs was made but further standardisation of the primers designed is required. This implies opportunities for functional characterisation of these mineral stress genes in Brassica and linseed for future use in molecular breeding.