The transcription factor OsbHLH035 mediates seed germination and enables seedling recovery from salt stress through ABA-dependent and ABA-independent pathways, respectively

Abstract

Background: Many transcription factors (TFs), such as those in the basic helix-loop-helix (bHLH) family, are important for regulating plant growth and plant responses to abiotic stress. The expression of OsbHLH035 is induced by drought and salinity. However, its functional role in rice growth, development, and the salt response is still unknown.

Results: The bHLH TF OsbHLH035 is a salt-induced gene that is primarily expressed in germinating seeds and seedlings. Stable expression of GFP-fused OsbHLH035 in rice transgenic plants revealed that this protein is predominantly localized to the nucleus. Osbhlh035 mutants show delayed seed germination, particularly under salt-stress conditions. In parallel, abscisic acid (ABA) contents are over-accumulated, and the expression of the ABA biosynthetic genes OsABA2 and OsAAO3 is upregulated; furthermore, compared with that in wild-type (WT) seedlings, the salt-induced expression of OsABA8ox1, an ABA catabolic gene, in germinating Osbhlh035 mutant seeds is downregulated. Moreover, Osbhlh035 mutant seedlings are unable to recover from salt-stress treatment. Consistently, sodium is over-accumulated in aerial tissues but slightly reduced in terrestrial tissues from Osbhlh035 seedlings after salt treatment. Additionally, the expression of the sodium transporters OsHKT1;3 and 1;5 is reduced in Osbhlh035 aerial and terrestrial tissues, respectively. Furthermore, genetic complementation can restore both the delayed seed germination and the impaired recovery of salt-treated Osbhlh035 seedlings to normal growth.

Conclusion: OsbHLH035 mediates seed germination and seedling recovery after salt stress relief through the ABA-dependent and ABA-independent activation of OsHKT pathways, respectively.

Keywords: ABA; OsHKT; Salt stress; Transcription factor; bHLH.

Fig. 1

OsbHLH035 expression is induced by salt and is abolished in the NG7221 line. a The structure of the OsbHLH035 gene and the retrotransposon insertion site. b OsbHLH035 expression patterns in aerial tissues from third-leaf-stage WT and NG7221 seedlings. The seedlings were grown on basal medium for 13 days and then transferred to basal medium containing 0 (CK) or 250 mM NaCl for an additional day. The primer positions and sequences are shown in (a) and Additional file 2: Table S1. Arabic numerals in (b) represent three independent biological replicates within each genotype

Fig. 2

The spatiotemporal expression of OsbHLH035 and the subcellular localization of GFP-OsbHLH035 in stable rice transformants. a The germinating seeds and post-germination-stage seedlings. The Z-scheme represents the staining order of the water-imbibed transgenic seeds, which were collected within 2 days. Em, Embryo; S, Scutellum; A, Aleurone layer; and En, Endosperm. b A third-leaf-stage seedling. c The presence of GFP-OsbHLH035 in the root tip. The upper panel is fluorescence image; the lower panel is overlaid with transmitted light image. Scale bars, 200 μm. d GFP-fused OsbHLH035 protein is localized with the DAPI, a nuclear affinity dye, in rice cells. Scale bars, 5 μm

Fig. 3

The delayed-germination phenotype of Osbhlh035 mutants is accompanied by the excess accumulation of endogenous ABA. a The germination of WT and Osbhlh035 seeds under normal and salt-treated conditions at 2 and 5 days after imbibition. Arabic numerals represent three independent biological replicates, and the seeds in each independent biological replicate were harvested from an individual rice plant. b The germination rates of WT and Osbhlh035 seeds under normal and salt-treated conditions. All germination rates represent the mean ± SD of two independent experiments performed with three independent biological replicates; 100 seeds were used in each independent biological replicate. c The endogenous ABA contents in both germinating WT and Osbhlh035 seeds grown under normal and salt-treated conditions on day 5. d The delayed-germination phenotype of Osbhlh035 mutants can be rescued by fluridone. The germination rates of WT and Osbhlh035 seeds under normal conditions at day 2 (upper panel) and salt-treated conditions at day 5 (lower panel). The seeds were grown on basal medium containing 0 (normal conditions, CK) or 250 mM NaCl (salt-treated conditions) supplemented with or without 50 μM fluridone. Samples for the ABA ELISA were harvested on day 5. Asterisks indicate significant differences in comparison with WT (*P < 0.05 and **P < 0.01) based on Student’s t-test

Fig. 4

The expression of ABA metabolic genes in both germinating WT and Osbhlh035 seeds. ABA over-accumulation in germinating Osbhlh035 seeds is due to the upregulation of the biosynthetic genes OsABA2 (a) and OsAAO3 (b) and abolition of salt-induced OsABAox1 (c) expression. The experimental materials and methods are the same as those described in Fig. 3. Samples for the q-PCR assay were harvested on day 5. Asterisks indicate significant differences in comparison with WT (*P < 0.05 and **P < 0.01) based on Student’s t-test

Fig. 5

A comparison of the growth patterns, salt responses, and ABA contents in both WT and Osbhlh035 seedlings under normal (CK) and salt-treated (100 mM NaCl) conditions. The seedlings were grown on basal medium for 7 days and then transferred to basal medium supplemented with 0 (CK) or 100 mM NaCl for an additional 7 days. a The growth patterns and salt responses of 14-day-old WT and Osbhlh035 aerial tissues. b The rates of water loss in 14-day-old WT and Osbhlh035 aerial tissues. c-d Characterization and quantification of root growth in both WT and Osbhlh035 seedlings. e The ABA contents in 14-day-old WT and Osbhlh035 seedlings. Scale bars in (a) and (c) represent 4 cm

Fig. 6

Effects of salinity on the Na⁺/K⁺ ratio and Na⁺ content in both WT and Osbhlh035 seedlings. a-b Na⁺/K⁺ ratios and Na⁺ contents in aerial and terrestrial tissues from both salt-treated WT and Osbhlh035 seedlings. c-d Na⁺/K⁺ ratios and Na⁺ contents in aerial and terrestrial tissues from both salt-relieved WT and Osbhlh035 seedlings. The seedlings were grown on basal medium for 11 days and then transferred to basal medium containing 0 (CK) or 100 mM NaCl (salt-treated conditions) for an additional 3 days. After day 14, the seedlings were returned to basal medium for 7 days (Recovery). Asterisks indicate significant differences in comparison to WT (*P < 0.05 and **P < 0.01) based on Student’s t-test

Fig. 7

Effects of salinity on the OsHKT1s expression profile in both WT and Osbhlh035 seedlings. a Quantification of OsHKT1;1, OsHKT1;3, and OsHKT1;5 mRNAs in aerial and terrestrial tissues from both salt-treated WT and Osbhlh035 seedlings. b Quantification of OsHKT1;3 and OsHKT1;5 mRNAs in aerial (left panel) and terrestrial (right panel) tissues, respectively, from both salt-relieved WT and Osbhlh035 seedlings. The seedlings were grown on basal medium for 11 days and then transferred to basal medium containing 0 (CK) or 100 mM NaCl (salt-treated conditions) for an additional 3 days. After day 14, the seedlings were returned to basal medium for 7 days (Recovery). Asterisks indicate significant differences in comparison to WT (*P < 0.05 and **P < 0.01) based on Student’s t-test

Fig. 8

Investigating growth responses, survival rate, and endogenous ABA contents in both WT and Osbhlh035 seedlings after recovery from salt stress. a-b The growth responses of aerial and terrestrial tissues in both WT and Osbhlh035 seedlings after relief from salt stress. c The primary root lengths of WT and Osbhlh035 seedlings after relief from salt stress. d The survival rate of WT and Osbhlh035 seedlings after relief from salt stress. All survival rates are shown as the mean ± SD of two independent experiments (n = 20 × 3 independent biological replicates within each genotype and/or experimental condition). e The endogenous ABA contents of WT and Osbhlh035 seedlings after recovery from salt stress. Scale bars in (a) and (b) represent 4 cm. The seedlings were grown on basal medium for 7 days and then transferred to basal medium containing 0 (control) or 100 mM NaCl (recovery) for an additional 7 days. After day 14, the seedlings were returned to basal medium for 7 days. Samples for the ABA ELISA were harvested on day 21. Asterisks indicate significant differences in comparison to WT (*P < 0.05 and **P < 0.01) based on Student’s t-test

Fig. 9

Examination of seed germination, endogenous ABA contents, and ABA metabolic gene expression in WT, Osbhlh035, and genetically complemented transformant rice. a-b The germination characteristics and rates of seeds from WT, Osbhlh035, and genetically complemented lines under normal and salt-treated conditions. Arabic numerals represent three independent biological replicates, which were harvested from three different rice plants per genotype. All germination rates are shown as the mean ± SD of two independent experiments; n = 50 × 3 biological replicates within each genotype and/or experimental condition. c The endogenous ABA contents of WT, Osbhlh035, and the genetically complemented transformant lines under normal and salt-treated conditions on day 4. d OsABA2, OsAAO3, and OsABA8ox1 expression in WT, Osbhlh035, and the genetically complemented transformant lines under normal and salt-treated conditions on day 4. The seeds were grown on basal medium supplemented with 0 (CK) or 250 mM NaCl. Samples for the ABA ELISA and q-PCR assays were harvested on day 4. Asterisks indicate significant differences in comparison to WT (*P < 0.05 and **P < 0.01) based on Student’s t-test

Fig. 10

Na⁺ content a of the aerial tissues and the expression of OsHKT1;3 b and OsHKT1;5 c in WT, Osbhlh035, and genetically complemented transformant lines. The seedlings were grown on basal medium for 11 days and then transferred to basal medium supplemented with 0 (CK) or 100 mM NaCl for an additional 3 days. Asterisks indicate significant differences in comparison to WT (*P < 0.05 and **P < 0.01) based on Student’s t-test

Fig. 11

Phenotypic characterization and ABA contents after recovery from salt treatment. a-d Root elongation, survival rates, and endogenous ABA contents in WT, Osbhlh035, and genetically complemented transformant lines after salt stress removal. The seedlings were grown on basal medium for 7 days and then transferred to basal medium supplemented with 0 (CK) or 100 mM NaCl (recovery) for an additional 7 days, after which the seedlings were returned to basal medium for 7 days. Samples for the ABA ELISA and q-PCR assays were harvested on day 21. Asterisks indicate significant differences in comparison to WT (*P < 0.05 and **P < 0.01) based on Student’s t-test