Sucrose nonfermenting-1-related protein kinase 1 regulates sheath-to-panicle transport of nonstructural carbohydrates during rice grain filling

Abstract

The remobilization of nonstructural carbohydrates (NSCs) reserved in rice (Oryza sativa) sheaths is essential for grain filling. This assimilate distribution between plant tissues and organs is determined by sucrose non-fermenting-1-related protein kinase 1 (SnRK1). However, the SnRK1-mediated mechanism regulating the sheath-to-panicle transport of NSCs in rice remains unknown. In this study, leaf cutting treatment was used to accelerate NSC transport in the rice sheaths. Accelerated NSC transport was accompanied by increased levels of OsSnRK1a mRNA expression, SnRK1a protein expression, catalytic subunit phosphorylation of SnRK1, and SnRK1 activity, indicating that SnRK1 activity plays an important role in sheath NSC transport. We also discovered that trehalose-6-phosphate, a signal of sucrose availability, slightly reduced SnRK1 activity in vitro. Since SnRK1 activity is mostly regulated by OsSnRK1a transcription in response to low sucrose content, we constructed an snrk1a mutant to verify the function of SnRK1 in NSC transport. NSCs accumulated in the sheaths of snrk1a mutant plants and resulted in a low seed setting rate and grain weight, verifying that SnRK1 activity is essential for NSC remobilization. Using phosphoproteomics and parallel reaction monitoring, we identified 20 SnRK1-dependent phosphosites that are involved in NSC transport. In addition, the SnRK1-mediated phosphorylation of the phosphosites directly affected starch degradation, sucrose metabolism, phloem transport, sugar transport across the tonoplast, and glycolysis in rice sheaths to promote NSC transport. Therefore, our findings reveal the importance, function, and possible regulatory mechanism of SnRK1 in the sheath-to-panicle transport of NSCs in rice.

Figure 1

The physiological function of rice sheath during grain filling. A–C, the dry weight (A), NSC content (B) and starch content (C) of -1/-2/-3/-4 sheaths during grain filling in Nipponbare. D–F, the dry weight (D), NSC content (E), and starch content (F) of -1/-2/-3/-4 internodes during grain filling in Nipponbare. Each value in (A and D) represents the mean ± sd of four replicates. Each value in (B, C, E, and F) represents the mean ± sd of three replicates. -1/-2/-3/-4 sheaths represent the first, second, third and fourth sheaths from the top, respectively. -1/-2/-3/-4 internodes represent the first, second, third and fourth internodes from the top, respectively. G–J, a mesophyll cell of the top of -2 sheath at 0 DAA (G), 5 DAA (H), 10 DAA (I), and 15 DAA (J). K, a chloroplast of the top part at 10 DAA. L–O, a mesophyll cell of the bottom of -2 sheath at 0 DAA (L), 5 DAA (M), 10 DAA (N), and 15 DAA (O). P, a chloroplast of the bottom part at 5 DAA. Abbreviation: Cl, chloroplast; CW, cell wall; SG, starch granules; Gr, grana.

Figure 2

Effects of LC treatment on grain filling and NSC remobilization in the sheaths. A, Schematic diagram of the LC treatment (three upper leaves were cut in half) at anthesis. B and C, The grain weight (B) and grain filling rate (C) of control (CK) and LC plants during grain filling. Each value in (B) represents the mean ± sd of six replicates. The grain filling rate was calculated using Richards’ equation (Richards, 1959). D, Starch staining of the sheaths from CK and LC plants during grain filling. E–H, The starch content (E), sucrose content (F), and relative expression levels of OsSUT1 (G) and OsSUT4 (H) in the sheaths of CK and LC plants during grain filling. Each value in (E–H) represents the mean ± sd of three replicates. Asterisks indicate significant differences between CK and LC plants at each time point by Student’s t test (B, E, F, G, and H): *P < 0.05, **P < 0.01.

Figure 3

Effects of LC treatment on SnRK1 activity in the sheaths during grain filling. A, SnRK1 activity in control (CK) and LC plants during grain filling. B, T6P contents in the sheaths of CK and LC plants during grain filling. C, The T6P content at each time point was plotted against the sucrose content. The Pearson correlation coefficients (r) for CK and LC plants are shown. Asterisks indicate significant correlations between T6P content and sucrose content using the data in Figures 2, F and 3, B: **P < 0.01, ***P < 0.001. D, SnRK1 activity in different tissues with 1 mM T6P at 10 DAA. E–G, Relative expression levels of OsSnRK1a (E), OSK24 (F), and OSK35 (G) in CK and LC plants during grain filling. H, Western blot analysis of SnRK1a protein abundance and phosphorylation level of the catalytic subunit in CK and LC plants at 0, 4, 10, and 15 DAA, with tubulin as internal control. Each value in (A) and (D) represents the mean ± sd of four replicates. Each value in (B) and (E–G) represents the mean ± sd of three replicates. Asterisks indicate significant differences between CK and LC plants at each time point by Student’s t test (A, B, E, F, and G): *P < 0.05, **P < 0.01. Asterisks indicate significant differences between −T6P and +T6P by Student’s t test (D): *P < 0.05, **P < 0.01.

Figure 4

Analysis of NSC remobilization in WT and snrk1a mutant plants during grain filling. A, The mutation site in the OsSnRK1a coding region. The triangle indicates the small guide RNA target. B, SnRK1 activity in the sheaths of snrk1a mutant plants at 0 and 4 DAA. Each value represents the mean ± sd of four replicates. C, Photo of the snrk1a mutant at 15 DAA. D, Starch staining of the sheaths at 0, 4, 10, 15, 20, 25, and 30 DAA. E and F, The starch (E) and sucrose (F) contents in the sheaths during grain filling. Each value represents the mean ± sd of three replicates. Asterisks indicate significant differences between WT and snrk1a plants at each time point by Student’s t test (B, E, and F): *P < 0.05, **P < 0.01.

Figure 5

Proteomic and phosphoproteomic profiling of the sheaths from WT and snrk1a mutant plants during grain filling. A, Experimental workflow of the analyses performed. B, Principal component analysis of all identified proteins and phosphosites per analysis. C, Number of DEPs and DESs in the sheaths at 0, 4, 10, and 15 DAA. The red and blue bars represent the upregulated and downregulated DEPs/DESs, respectively. D, Venn diagram of the DESs among the four comparisons. E, Cluster analysis of the 599 DESs identified. The color scale represents the relative phosphorylation level, from low (green) to high (red). F, The phosphorylation motifs identified using Motif-X algorithm within the putative SnRK1 targets and/or SnRK1-dependent phosphosites. G, Predicted kinase activity of MAPKs, CPKs, and TOR. The color scale represents the kinase activity score, from low (blue) to high (red).

Figure 6

Phosphoproteomic analysis of SnRK1-dependent phosphosites involved in the sheath transport of NSCs. A, Overview of the analysis performed. B, Module–trait (starch content) relationships and their corresponding P-values. The eight-colored part (left) shows the seven PPMs and one unclustered module. The color scale (right) represents the module–trait (starch content) correlations, from low (green) to high (red). C, Functional enrichment analysis of the PPMs using the GO and KEGG databases.

Figure 7

PRM assay of 20 phosphosites involved in the sheath transport of NSCs at 6 DAA. PRM assay results showing the associated pathways of the 20 phosphosites identified in the sheaths of control (CK) and LC treatment plants at 6 DAA. Each value represents the mean of three replicates. For more information, please refer to Supplemental Table S9.

Figure 8

A model for the SnRK1-mediated regulation of NSC sheath-to-panicle remobilization in response to sucrose availability. NSC transport in sheaths is regulated by sucrose availability and SnRK1 activity to ensure sufficient assimilation for grain filling. If the source leaves are sufficient for grain development, starch accumulation occurs in the rice sheaths. In case of reduced photosynthetic leaf area or when the peak of grain filling is reached, the leaves will not be sufficient for grain development. Hence, the sucrose stored in the sheaths is transported to the panicles, resulting in a low sucrose level in the sheaths. The low sucrose content induces an increase in OsSnRK1a expression and reduces the T6P mediated inhibition of SnRK1. Elevated SnRK1 activity subsequently promotes the sheath to panicle transport of NSCs through the regulation of starch degradation, sugar transport across the tonoplast, sucrose metabolism, phloem transport, and glycolysis via phosphorylation. The orange arrows represent sucrose flow (left). The red and blue represent upregulation and downregulation, respectively (right).