Identification and expression analysis of miRNAs and elucidation of their role in salt tolerance in rice varieties susceptible and tolerant to salinity

Abstract

Soil salinization is a serious problem for cultivation of rice, as among cereals rice is the most salt sensitive crop, and more than 40% of the total agricultural land amounting to approximately 80 million ha the world over is salt affected. Salinity affects a plant in a varieties of ways, including ion toxicity, osmotic stress and oxidative damage. Since miRNAs occupy the top place in biochemical events determining a trait, understanding their role in salt tolerance is highly desirable, which may allow introduction of the trait in the rice cultivars of choice through biotechnological interventions. High throughput sequencing of sRNAs in the root and shoot tissues of the seedlings of the control and NaCl treated Pokkali, a salt-tolerant rice variety, identified 75 conserved miRNAs and mapped 200 sRNAs to the rice genome as novel miRNAs. Expression of nine novel miRNAs and two conserved miRNAs were confirmed by Northern blotting. Several of both conserved and novel miRNAs that expressed differentially in root and/or shoot tissues targeted transcription factors like AP2/EREBP domain protein, ARF, NAC, MYB, NF-YA, HD-Zip III, TCP and SBP reported to be involved in salt tolerance or in abiotic stress tolerance in general. Most of the novel miRNAs expressed in the salt tolerant wild rice Oryza coarctata, suggesting conservation of miRNAs in taxonomically related species. One of the novel miRNAs, osa-miR12477, also targeted L-ascorbate oxidase (LAO), indicating build-up of oxidative stress in the plant upon salt treatment, which was confirmed by DAB staining. Thus, salt tolerance might involve miRNA-mediated regulation of 1) cellular abundance of the hormone signaling components like EREBP and ARF, 2) synthesis of abiotic stress related transcription factors, and 3) antioxidative component like LAO for mitigation of oxidative damage. The study clearly indicated importance of osa-miR12477 regulated expression of LAO in salt tolerance in the plant.

Fig 1. Northern blots showing expression of two conserved miRNAs in root (R) and shoot (S) tissues of control (C) and 256 mM NaCl treated (T) seedlings of O. sativa cv. Pokkali (salt-tolerant) and O. sativa cv. Badami (salt-sensitive) on 9^th day of germination.

The right panel shows the relative expression of the individual miRNAs in the root/shoot tissues of the treated seedlings over control ones, as obtained by densitometric analysis of the band intensity considering the band intensity of U6 for normalization.

Fig 2. Northern blots showing expression of the novel miRNAs.

Other details as in Fig 1.

Fig 3. Northern blot showing expression of the novel miRNAs in the shoot tissue of control (C) and 256 mM NaCl treated (T) Oryza coarctata (OC) and Suaeda maritima (SM).

NaCl treatment was for 9 h. U6 served as internal control. The right panel shows the relative expression of the miRNAs in the treated over control after normalization of the band intensity with U6.

Fig 4. The cleavage points of L-ascorbate oxidase (LAO) and monocopper oxidase (MCO) targeted by the novel miRNA osa-miR12477, and of Myb targeted by the novel miRNAs osa-miR12482.

The cleavage points, indicated by arrows, were obtained by cloning and sequencing of the 5’RACE PCR products. The numbers against each arrow indicate the number of clones that were having cleavage point, i.e. the insert ligated to the 5’ adaptor out of the total number of the clones of the PCR products sequenced.

Fig 5. Dual-luciferase assay for the validation of the target cleavage.

The target sequence of the miRNAs in the three gene products, i.e. L-Ascorbate oxidase (LAO), monocopper oxidase (MCO), and Myb were introduced individually in the 3’UTR of luciferase (Addgene ID 55206). These constructs served as 3’UTR sensor. Construct was also made for overexpression of osa-miR12477 and osa-miR12482 by introducing their hairpin region flanked with 100 bp upstream and downstream sequences separately into Addgene ID 55208 vector. These were termed miRNA overexpressor. The 3’UTR sensor and miRNA overexpressor were then individually introduced into A. tumefaciens strain LBA4404. Nine leaves in taking three plants were infiltrated with equal volumes of miRNA overexpressor and 3’UTR sensor. Leaves were also infiltrated only with 3’UTR sensor, referred as (-) MO. These leaves were collected after 48 h and Renilla (R) and Firefly (F) luciferase assays were performed. The ratio of F-Luc/R-Luc activities for the individual 3’UTR sensor and miRNA overexpressor pairs were calculated and presented (a). Parallelly RNA was also extracted from the infiltrated leaves. RT-qPCR was performed for R-luciferase and F-luciferase using primers against these transcripts to see the relative expression of F-luciferase using the expression of R-luciferase as reference (b). The data are mean ± sd of assays in nine individual samples. The asterisk, ** or *** marked against the individual genes indicates that their activities/expressions in the leaves infiltrated with both 3’UTR sensor and miRNA overexpressor for the individual gene and miRNA pairs were significantly less than in the leaves infiltrated only with 3’UTR sensor at p ≤ 0.01 and p ≤ 0.001, respectively.

Fig 6. Changes in expression of LAO and Myb in shoot (S) and root (R) tissues of O. sativa cv. Pokkali and O. sativa cv. Badami upon exposure to 256 mM NaCl for 9 h on the 9^th day of germination.

The expression was measured by RT-qPCR and expressed as relative value in the treated samples over the control ones considering actin as the reference gene. LAO- L-Ascorbate oxidase, Myb- Myb transcription factor.

Fig 7. Influence of NaCl treatment on accumulation of H₂O₂ (ROS) in the leaves of salt-tolerant O. sativa cv. Pokkali and salt-sensitive O. sativa cv. Badami.

The leaves of the control seedlings and those treated with 256 mM NaCl for 9 h on the 9^th day of germination were harvested and processed for DAB staining for the detection of ROS accumulation. Three leaves each from the control and NaCl treated seedlings of both Pokkali and Badami were considered for DAB staining.

Fig 8. In situ PCR for the localization and expression studies of osa-miR12477.

Paraffin embedded leaves of the control (C) and 256 mM NaCl treated (T) Pokkali seedlings were sectioned (60 μm) on a microtome, processed to remove the paraffin, osa-miR12477 was reverse transcribed using miRNA specific primer and amplified with miRNA specific stem-loop forward primer and universal reverse primer using high fidelity Taq polymerase with dig-11-dUTP (Roche). The section was probed with anti-DIG-AP-Fab fragment and the miRNA expression was visualized using BM-purple substrate (Roche) on a light microscope. The leaf sections of both control (C) and NaCl treated (T) seedlings were processed without (A) and with (B) osa-miR12477 specific stem-loop primer.