A cotton miRNA is involved in regulation of plant response to salt stress

Abstract

The present study functionally identified a new microRNA (microRNA ovual line 5, miRNVL5) with its target gene GhCHR from cotton (Gossypium hirsutum). The sequence of miRNVL5 precursor is 104 nt long, with a well developed secondary structure. GhCHR contains two DC1 and three PHD Cys/His-rich domains, suggesting that GhCHR encodes a zinc-finger domain-containing transcription factor. miRNVL5 and GhCHR express at various developmental stages of cotton. Under salt stress (50-400 mM NaCl), miRNVL5 expression was repressed, with concomitant high expression of GhCHR in cotton seedlings. Ectopic expression of GhCHR in Arabidopsis conferred salt stress tolerance by reducing Na(+) accumulation in plants and improving primary root growth and biomass. Interestingly, Arabidopsis constitutively expressing miRNVL5 showed hypersensitivity to salt stress. A GhCHR orthorlous gene At2g44380 from Arabidopsis that can be cleaved by miRNVL5 was identified by degradome sequencing, but no confidential miRNVL5 homologs in Arabidopsis have been identified. Microarray analysis of miRNVL5 transgenic Arabidopsis showed six downstream genes (CBF1, CBF2, CBF3, ERF4, AT3G22920, and AT3G49200), which were induced by salt stress in wild-type but repressed in miRNVL5-expressing Arabidopsis. These results indicate that miRNVL5 is involved in regulation of plant response to salt stress.

Figure 1. Basic information on miRNVL5 and GhCHR from cotton (Gossypium hirsutum).

(A) Mature (in red) and precursor sequences and the stem-loop structure of miRNVL5. (B) The GhCHR sequence with nucleotides was cleaved by miRNVL5 using 5′-RACE. (C) cDNA structure of GhCHR. (D) Predicted GhCHR protein structure with PHD and C1 domains. (E) Dendrogram showing the relationship among genes encoding PHD-type proteins in cotton and other plant species. (F) Dendrogram showing PHD-type proteins between cotton and Arabidopsis only.

Figure 2. Expression of miRNVL5, pri-miRNVL5 and GhCHR in different tissues of cotton with or without salt treatment.

(A,D,G) Genes from ovules of −2–20 days post anthesis (DPA). (B,E,H) Genes from developing fibers of 10, 15 and 20 DPA. (C,F,I) genes from other tissues. (J–M) Two week-old cotton seedlings were treated with NaCl (0–400 mM) (J,K) for 1 h, or with 250 mM NaCl for 0–6 h (L,M). After that, total RNA was extracted and analyzed by real-time PCR. Vertical bars represent SD of the mean with three replicates. Asterisks indicate that mean values are significantly different between the treatment and control (p < 0.05).

Figure 3

Seed germination and seedling growth of transgenic Arabidopsis over-expressing miRNVL5 (35::miRNVL5) (A,D,G,J,M), miRNVL5-resistant version (35S::mGhCHR)(B,E,H,K,N) and GhCHR (35S::GhCHR)(C,F,I,L,O) under the salt stress. (A–C) Seeds were germinated on MS medium containing 100 mM NaCl. Germination rates were measured during one week. (D–F) Cotyledon greening and expansion of 7 day-old seedlings with 50–150 mM NaCl. (G–I) Fresh weight of 7 day-old seedlings exposed to 50–150 mM NaCl. (J–K) The elongation of primary root of 7 day-old seedlings exposed to 50–150 mM NaCl. M-O: phenotype of primary root growth of 7 day-old seedlings exposed to 50–150 mM NaCl. Vertical bars represent SD of the mean with three replicates. Asterisks indicate that mean values are significantly different between the transgenic plants and wild-type (WT) (p < 0.05).

Figure 4. Sodium (Na⁺)/potassium (K⁺) accumulation and expression of SOS1, NHX1, AVP1 and P5CS1 in transgenic and wild-type (WT) plants exposed to NaCl.

(A,C) Na⁺ in shoots and roots. (B,D) K⁺ in shoots and roots of three week-old WT, 35S::miRNVLU5 and 35S::mGhCHR transgenic plants exposed to 200 mM NaCl exposure for 3 d. (E,F) K⁺/Na⁺ ratio in WT and the transgenic plants under −NaCl (E,F) and +NaCl (F) Condition. (G–J) qRT-PCR analysis of the indicated gene expression. Three week-old seedlings were exposed to 200 mM NaCl for 1 h. Vertical bars represent SD of the mean with three replicates. Asterisks indicate that mean values are significantly different between the transgenic plants and WT (p < 0.05).

Figure 5. Identification of miRNVL5 target from Arabidopsis through degradome sequencing.

(A) The red line with a dot indicates the significant signature. The arrow indicates the signature produced by miRNA-directed cleavage. (B) Target for miRNVL5 identified from degradome of 35S::miRNVL5 Arabidopsis exposed to minus salt (miR5 − S) and plus salt (miR5 + S). Three week-old Arabidopsis seedlings were treated with 0 (control) and 200 mM NaCl for 1 h and the treated seedlings were used for degradome sequencing and qRT-PCR.

Figure 6. Differentially expressed genes in Arabidopsis wild-type (Col-0) and 35S::miRNVL5 seedlings under salt stress.

Three weeks-old Arabidopsis seedlings were treated with 200 mM NaCl for 1 h. (A) Venn diagram showing up- and down-regulated genes in Col-0 and 35S::miRNVL5 seedlings under salt stress. (B) Venn diagram showing up- and down-regulated genes in 35S::miRNVL5 seedlings relative to Col-0 grown on the control (0 mM) and 200 mM NaCl media.

Figure 7. Co-expression, network and Gene Orthology (GO) analysis of salt-inducible genes in Arabidopsis.

(A) Differentially expression profiles of salt-responsive genes that are co-expressed with AT2g44380 in Arabidopsis treated with 100 mM NaCl for 0–32 h. Hierarchial clustering display performed using Euclidean distance according to expression pattern with the MeV software. The intensities of the color represent the relative magnitude of fold changes in log values. The raw transcriptional data that the heatmap used were obtained from Li et al. (2009) and normalized using RMA algorithm (Dinneny, et al., 2008) with Expression Console software (Affymetrix Technologies). (B) The hypothetic network under salt stress. (C) Gene Orthology analysis.

Figure 8. The model for cotton miRNVL5 regulating its target genes GhCHR (cotton) and At2g44380 (Arabidopsis).

The schematic model reflects that transformation of miRNVL5 into Arabidopsis results in decrease in K/Na ratio and seedling sensitivity to salt stress possibly through repressing At2g44380, and several other downstream genes CBF1, CBF2, CBF3, ERF4, AT3G22920, AT3G49200 and others.