The MKKK62-MKK3-MAPK7/14 module negatively regulates seed dormancy in rice

Abstract

Background: Seed dormancy directly affects the phenotype of pre-harvest sprouting, and ultimately affects the quality and yield of rice seeds. Although many genes controlling seed dormancy have been cloned from cereals, the regulatory mechanisms controlling this process are complex, and much remains unknown. The MAPK cascade is involved in many signal transduction pathways. Recently, MKK3 has been reported to be involved in the regulation of seed dormancy, but its mechanism of action is unclear.

Results: We found that MKKK62-overexpressing rice lines (OE) lost seed dormancy. Further analyses showed that the abscisic acid (ABA) sensitivity of OE lines was decreased. In yeast two-hybrid experiments, MKKK62 interacted with MKK3, and MKK3 interacted with MAPK7 and MAPK14. Knock-out experiments confirmed that MKK3, MAPK7, and MAPK14 were involved in the regulation of seed dormancy. The OE lines showed decreased transcript levels of OsMFT, a homolog of a gene that controls seed dormancy in wheat. The up-regulation of OsMFT in MKK3-knockout lines (OE/mkk3) and MAPK7/14-knockout lines (OE/mapk7/mapk14) indicated that the MKKK62-MKK3-MAPK7/MAPK14 system controlled seed dormancy by regulating the transcription of OsMFT.

Conclusion: Our results showed that MKKK62 negatively controls seed dormancy in rice, and that during the germination stage and the late stage of seed maturation, ABA sensitivity and OsMFT transcription are negatively controlled by MKKK62. Our results have clarified the entire MAPK cascade controlling seed dormancy in rice. Together, these results indicate that protein modification by phosphorylation plays a key role in controlling seed dormancy.

Keywords: Dormancy; MAPK cascade; Pre-harvest sprouting; Rice (Oryza sativa L.).

Fig. 1

Overexpression of MKKK62 in ZH11 resulting in PHS. a, Germination phenotype of WT and OE panicles. At 30 DAH, panicles of OE1, OE2, OE3, and WT were sampled for germination tests. Seeds from OE lines germinated at 3 DAI. b, Germination percentage of seeds from OE panicles sampled at indicated times. Seeds were harvested at indicated times from OE plants for germination test. Germination percentage was calculated at 2 DAI. Values represent the mean ± SD of three biological replicates. c, Relative expression level of MKKK62 in seeds of MKKK62-overexpression (OE) lines at 23 DAH. At 23 DAH, gene transcript levels in seeds of OE1, OE2, OE3, and WT were analyzed by real-time PCR

Fig. 2

ABA and GAs contents in seeds of OE and WT lines. At 23 DAH, seeds from three OE lines and WT were harvested and stored in liquid nitrogen until analysis. Values represent the mean ± SD of at least three replicates. Student’s t-tests were used to generate P values

Fig. 3

Decreased ABA sensitivity in OE lines. a, Growth performance of OE lines and WT on 1%(w/v) agar without or with 10 μM ABA. In mock treatment, agar contained equal amount of solvent. b, Length of shoot and root of OE lines after 3 days of treatment. Shoot and root lengths were measured after 3 days of culture in the light at 30 °C.Values represent the mean ± SD of three OE lines (ten replicates/line)

Fig. 4

Interactions between indicated proteins. a, Interactions between indicated proteins were investigated by Y2H. Plus sign, positive control; minus sign, negative control; QD + AbA, quadruple-dropout medium lacking Ade, His, Leu and Trp containing 200 ng/mL AbA; DD, double-dropout medium lacking Leu and Trp. b, c, d, and e, pull-down results of four pairs of proteins: MKKK62-MKK3, MKKK62-MKK10–2, MKK3-MAPK7, and MKK3-MAPK14, respectively. Binding of GST- and His-fusion proteins was tested by immunoblotting using anti-His after pull-down with GST-fusion protein. GST was used as negative control. Expression of fusion proteins was detected using antibody to the tag. “Input” indicated the detection result of the fusion proteins with anti-His or anti-GST antibody; “Pull-down” indicated the detection result of His-fusion protein binding to GST-fusion protein with anti-His antibody. Arrow indicates the band of the target protein

Fig. 5

Germination phenotype of knockout progenies. a, At 23 DAH, seeds were collected from different knockout progenies and germination percentage was calculated at 2 DAI. b, At 30 DAH, seeds were collected from homozygous knockout progenies and germination percentage was calculated at 5 DAI. c, Time course of germination of homozygous OE/mkk3 and OE/mapk7/mapk14 seeds after storage at 37 °C for 1 month. mkk3, MKK3 knockout plant; mapk7, MAPK7 knockout plant; mapk14, MAPK14 knockout plant; P, homozygous positive; H, heterozygous; Number in parentheses indicates number of knockout lines. Values are mean ± SD

Fig. 6

Relative transcript levels of OsMFT in different transgenic materials. Transcript level of OsMFT was analyzed by real-time PCR using seeds collected at 23 DAH. Values shown are the mean ± SD of triplicate independent biological samples (n = 3). Transcript level was normalized to that of OsMFT in WT. Student’s t-tests were used to generate P values

Fig. 7

Model of regulation of seed dormancy in rice. Arrows indicate transcriptional activation, flat-ended arrows indicate transcriptional repression. Arrows with ℗ indicate activation by phosphorylation. Question mark indicates unknown part