NRL3 Interacts with OsK4 to Regulate Heading Date in Rice

doi:10.1016/j.rsci.2022.01.010

Abstract

Abstract:

NRL3 is essential for the growth and development of rice leaves. In this study, we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions. The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3 interacts with OsK4, a Snf1-related kinase. OsK4 localized to the nucleus and expressed in various rice tissues. The rhythmic expression pattern of OsK4 was similar to NRL3 under long daylight and short daylight conditions. Knock-out mutants of OsK4 exhibited early heading under long daylight conditions, indicating that it acts as a negative regulator of heading date in rice. Interestingly, the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions, suggesting that OsK4 functions downstream of NRL3. Moreover, both NRL3 and OsK4 controlled heading date through regulating the expression of Hd3a and RFT1 genes. These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.

Key words: Oryza sativa, heading date, NRL3 gene, OsK4 gene, negative regulator, loss function

Chen Wei, Cai Yicong, Shakeel Ahmad, Wang Yakun, An Ruihu, Tang Shengjia, Guo Naihui, Wei Xiangjin, Tang Shaoqing, Shao Gaoneng, Jiao Guiai, Xie Lihong, Hu Shikai, Sheng Zhonghua, Hu Peisong. NRL3 Interacts with OsK4 to Regulate Heading Date in Rice[J]. Rice Science, 2022, 29(3): 237-246.

Figures/Tables 6

Fig. 1. NRL3 positively regulates heading date under long-day (LD) and short-day (SD) conditions. A, Late heading phenotypes of nrl3 in the field. Scale bar, 20 cm. WT, Wild type (Zhongjiazao 17). B, Heading dates of WT and nrl3 under natural long-day (NLD) and natural short-day (NSD) conditions. Data are Mean ± SD (n = 15). C and D, WT and nrl3 had similar leaf emergence rates under LD (14 h light/10 h dark) and SD (10 h light/14 h dark) conditions. Data are Mean ± SD (n = 10). E, Phenotypes of NRL3 overexpression lines (OE-1 and OE-2) in nrl3 background. Scale bar, 20 cm. F, Expression levels of NRL3 in WT, nrl3 and overexpression lines (OE-1 and OE-2). RNA was extracted from the flag leaves of 60-day-old plants under NLD conditions. The rice Actin gene was used as the internal control. Data are Mean ± SD from three individual replicates. G and H, Heading dates of WT, nrl3 and overexpression lines (OE-1 and OE-2) under NLD and NSD conditions. Data are Mean ± SD (n = 15). Asterisks indicate statistical significance as determined by the Student’s t-test (**, P < 0.01).

Fig. 2. Expression patterns of NRL3 (A and B), RFT1 (C and D), Hd3a (E and F), Ehd1 (G and H) and Hd1 (I and J) in wild type (WT) Zhongjiazao 17 and nrl3 mutant under long-day (LD) and short-day (SD) conditions. RNA was extracted from the leaves of 20-day-old WT and nrl3 under SD and LD conditions. The rice Actin gene was used as the internal control. Data are Mean ± SD from three individual replicates. Open bars indicate light periods and filled bars indicate dark periods.

Fig. 3. NRL3 forms a complex with OsK4. A, Interaction between NRL3 and OsK4 in yeast cells. pGBKT7-53 (53) and pGADT7-T (T) were used as positive controls, and pGBKT7 (BD) and pGADT7 (AD) were used as negative controls. SD/-H-A, SD/-Trp-Ade; SD/-H-A-T-L, SD/-His-Ade-Trp-Leu. B, Bimolecular fluorescence complementation (BiFC) analysis of interaction of NRL3 with OsK4 in Nicotiana benthamiana epidermal cells. pSPYNE (YN) and pSPYCE (YC) were used as negative controls. Scale bars, 20 μm. YFP, Yellow fluorescent protein.

Fig. 4. Expression pattern analysis of OsK4. A, Temporal and spatial expression pattern of OsK4. Seed-5d and seed-10d correspond to developing endosperm at 5 and 10 d after fertilization, respectively. Rice Actin gene was used as the internal control. B, Subcellular location of OsK4-GFP fusion protein in rice protoplasts. Free- GFP (green fluorescent protein) was used as the control, and D53-mCherry as a nuclear marker. Scale bars, 10 μm. C and D, Rhythmic expression patterns of OsK4 under long-day and short-day conditions. Open bars indicate light periods, and filled bars indicate dark periods. RNA was extracted from the leaves of 20-day-old wild type (Zhongjiazao 17) under long-day and short-day conditions. The rice Actin gene was used as an internal control.Data are Mean ± SD from three individual replicates.

Fig. 5. Phenotypes of OsK4 knock-out transgenic plants and expression patterns of heading date relative genes in ko-osk4 mutants. A, Phenotypes of ko-osk4 plants in field. WT, Wild type (Nipponbare). Scale bar, 20 cm. B and C, Heading date of ko-osk4 plants under natural long-day (NLD) and natural short-day (NSD) conditions. D?G, mRNA levels of NRL3, Hd3a, RFT1 and Ehd1 in WT and ko-osk4. Rice Actin gene was used as the internal control. RNA was extracted from the flag leaves of 60-day-old WT and ko-osk4 mutants under NLD conditions.Data are Mean ± SD from three individual replicates. Asterisks indicated statistical significance as determined by the Student’s t-test (**, P < 0.01).

Fig. 6. Knock-out OsK4 rescues late heading phenotype of nrl3. A, Heading phenotypes of wild type (WT, Zhongjiazao 17), nrl3 mutant and nrl3/osk4 double mutant. Scale bar, 20 cm. Red arrows indicate heading. B, Heading dates of WT, nrl3 and nrl3/osk4 under natural long-day (NLD) conditions. C, Expression levels of OsK4 in WT, nrl3 and nrl3/osk4. RNA was extracted from the leaves of 60-day-old plants under NLD conditions. The rice Actin gene was used as the internal control. D, Expression levels of OsK4 in WT, nrl3 and OE-NRL3. RNA was extracted from the leaves of 60-day-old plants under NLD conditions. The rice Actin gene was used as the internal control. E, Protein levels of OsK4 in WT, nrl3 and nrl3/osk4 by immunoblot analysis. β-ACTIN was used as the internal control. F, OsK4 protein abundance in WT, nrl3 and nrl3/osk4, quantified by ImageJ program. G, Protein levels of OsK4 in WT, nrl3 and OE-NRL3 by immunoblot analysis. β-ACTIN was used as the internal control. H, OsK4 protein abundance in WT, nrl3 and OE-NRL3, quantified by ImageJ program. I, Assay of OsK4 protein stability in vitro. GST-OsK4 proteins were purified from E. coli and incubated with the equal total extracts of WT or nrl3 seedlings in the presence or absence of MG132. GST-OsK4 proteins were detected by GST antibody. J, Quantitative assay of OsK4 protein levels. Data are Mean ± SD from three individual replicates. Asterisks indicate statistical significance as determined by the Student’s t-test (**, P < 0.01).

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