Grain setting defect1 (GSD1) function in rice depends on S-acylation and interacts with actin 1 (OsACT1) at its C-terminal

Abstract

Grain setting defect1 (GSD1), a plant-specific remorin protein specifically localized at the plasma membrane (PM) and plasmodesmata of phloem companion cells, affects grain setting in rice through regulating the transport of photoassimilates. Here, we show new evidence demonstrating that GSD1 is localized at the cytoplasmic face of the PM and a stretch of 45 amino acid residues at its C-terminal is required for its localization. Association with the PM is mediated by S-acylation of cysteine residues Cys-524 and Cys-527, in a sequence of 45 amino acid residues essential for GSD1 function in rice. Furthermore, the coiled-coil domain in GSD1 is necessary for sufficient interaction with OsACT1. Together, these results reveal that GSD1 attaches to the PM through S-acylation and interacts with OsACT1 through its coiled-coil domain structure to regulate plasmodesmata conductance for photoassimilate transport in rice.

Keywords: S-acylation; plasma membrane; plasmodesmata; remorin; rice.

FIGURE 1

A stretch of 45 amino acid residues at the C-terminal is sufficient for grain setting defect 1 (GSD1) association with PM. (A) Schematic representation of full-length and truncated GSD1 fragments. CCD, coiled-coil domain. (B) Full-length and truncated GSD1 fragments were fused with GFP and expressed in tobacco leaves. Images show that GSD1C1 and GSD1C3 are localized specifically on PM. Bars = 50 μm. (C) Western blot detection of GFP-GSD1C3, GFP-GSD1C4, and GFP-GSD1C5 in soluble and membrane fractions of the transformed tobacco leaves. T, total; S, soluble; MF, membrane fractions.

FIGURE 2

Analysis of GSD1 association with PM. Coexpression of GFP-GSD1 with Golgi marker GmMan1:mCherry or COPII-mediated ER-Golgi transport regulator mutant Sar1H74L-mCherry in tobacco leaves. Images show that GSD1 trafficking to PM is unaffected by BFA treatment, while subcellular localization of GmMan1:mCherry is changed. Additionally, GSD1 trafficking to PM are not affected when coexpressed with mutant Sar1H74L-mCherry. Bars = 50 μm.

FIGURE 3

Grain setting defect 1 association with PM through S-acylation. (A) Alignment of the GSD1 C-terminal sequences with its homologous proteins. The C-terminal sequences contain a coiled-coil domain but vary at the end amino acid residues. Asterisk indicates possibly S-acylated cysteine residues. (B) Biotin switch assays of S-acylation state of GSD1 in rice membrane fractions. S-acylated GSD1 is detected by Western blotting in the elution fraction of the hydroxylamine-treated sample but not detected in the untreated sample. Hyd+, with hydroxylamine; Hyd-, without hydroxylamine. (C) Comparison of the GSD1 S-acylation with or without 2-bromopalmitate (2-BP) (an S-acylation inhibitor) treatment. 2-bromopalmitate treatment substantially reduced the amount of S-acylated GSD1. (D) Western blotting of GSD1 in sucrose density gradient fractions of membrane proteins from rice with GSD1 specific antibodies. GSD1 is predominantly detected in the upper fractions (fractions: 4–6), whereas 2-BP treatment substantially reduces GSD1 in these fractions. (E) Confocal microscopic observation of the subcellular localization of GFP-GSD1 in tobacco leaves under 2-BP treatment or mock solution. Arrowhead denotes GFP fluorescent in nucleus. Bars = 50 μm.

FIGURE 4

The cysteine residues Cys-524 and Cys-527 are essential for GSD1 PM targeting and S-acylation. (A) Schematic representation of multiple mutations of cysteine residues in the GSD1 C-terminal sequence. (B) Subcellular localization of GSD1 multiple mutations in tobacco leaves. The quadruple mutations M6 and M7 displayed a specific localization on PM. Bars = 50 μm.

FIGURE 5

Analysis of the GSD1 domain for binding to OsACT1. (A) GSD1 domains (GSD1N, GSD1C1, or GSD1C2, shown in Figure 2A) are examined for interactions with OsACT1 using bimolecular fluorescence complementation (BiFC) assay. GSD1N: GSD1 N-terminal fragment; GSD1C1, GSD1 C-terminal fragment; GSD1C2: actin-binding domain. Bars = 50 μm. (B) and (C) Interaction of GSD1C1-Myc (B) or GSD1C2-Myc (C) with OsACT1-Flag is detected by Co-immunoprecipitation (Co-IP). GSD1-Myc and OsACT1-FLAG were individually expressed or combinationally co-expressed in tobacco leaves. Total protein extract was immunoprecipitated with anti-Myc antibodies coupled agarose beads or anti-Flag antibodies coupled agarose beads. Proteins from crude lysate and immunoprecipitation were detected with anti-Myc antibodies and anti-Flag antibodies, respectively.

FIGURE 6

S-acylation is crucial for GSD1 regulation of the PD conductance. (A) and (B) Statistical analyses of grain setting rate (A) and spikelet number (B) in WT, GSD1 cysteine mutants (GSD1M3OX, GSD1M6OX, GSD1M7OX) and GSD1 deletion mutant (GSD1^ΔCOX) transgenic plants. Values are means ± SE of 15 independent plants. (C) Quantitative RT-PCR analyses of the GSD1 expression in panicles at flowering stage. Results are means ± SE of three individual samples. (D–F) Measurement of the ¹³C-labeled sucrose in flag leaf blades (D), flag leaf sheaths (E) and phloem exudate (F) after flag leaf blade photosynthesis fed with ¹³CO₂.