Lack of the α1,3-Fucosyltransferase Gene (Osfuct) Affects Anther Development and Pollen Viability in Rice

Abstract

N-linked glycosylation is one of the key post-translational modifications. α1,3-Fucosyltransferase (OsFucT) is responsible for transferring α1,3-linked fucose residues to the glycoprotein N-glycan in plants. We characterized an Osfuct mutant that displayed pleiotropic developmental defects, such as impaired anther and pollen development, diminished growth, shorter plant height, fewer tillers, and shorter panicle length and internodes under field conditions. In addition, the anthers were curved, the pollen grains were shriveled, and pollen viability and pollen number per anther decreased dramatically in the mutant. Matrix-assisted laser desorption/ionization time-of-flight analyses of the N-glycans revealed that α1,3-fucose was lacking in the N-glycan structure of the mutant. Mutant complementation revealed that the phenotype was caused by loss of Osfuct function. Transcriptome profiling also showed that several genes essential for plant developmental processes were significantly altered in the mutant, including protein kinases, transcription factors, genes involved in metabolism, genes related to protein synthesis, and hypothetical proteins. Moreover, the mutant exhibited sensitivity to an increased concentration of salt. This study facilitates a further understanding of the function of genes mediating N-glycan modification and anther and pollen development in rice.

Keywords: N-glycan; anther; development; microarray; pollen; viability; α1,3-fucosyltransferase.

Figure 1

Identification and genotyping of the T-DNA insertion Osfuct mutant in rice (Oryza sativa). (A) Schematic representation of the genomic structure of the Osfuct and T-DNA insertion sites. Black-lined box represents promotor, while the yellow box indicates exons, and the white box represents the introns. Locations of the T-DNA insertion and direction of the left and right borders are indicated by an inverted triangle. Gene-specific (P1, P2, and P3) and T-DNA border sequence primers (P4) were used for genotyping and gene-specific primers (P5 and P6) were used for reverse transcription polymerase chain reaction (RT-PCR) analysis as indicated by the black arrowhead. (B) Yellow box with black border represents the full length of the cDNA (1542 bp). (C) SMART annotation for the OsFucT protein. Magenta box indicates the low complexity region, blue box shows transmembrane domain, and the green box represents the conserved glycosyltransferase domain (220–398 amino acids). (D) Phylogenetic analysis of OsFucT with other glycosyltrasnferase proteins. (E) Genotyping of homozygote (HM) and heterozygote (HT) plants to confirm the T-DNA insert. The upper lane PCR products were amplified using gene-specific primers P1 and P3 (1.2 kb), whereas the lower lane PCR products were amplified using the gene-specific primer P3 and T-DNA border sequence primer P4 (0.6 kb). (F) Expression analysis of Osfuct in Dongjin (DJ), HM and HT lines was done using RT-PCR. The expression level of actin1 was used as a loading control and gene specific primers (P5 and P6) were used for RT-PCR analysis.

Figure 2

Morphological and reproductive phenotype of the Osfuct HM mutant and HT plant compared to Dongjin (DJ) under field conditions. (A) Comparison of plant height, culm length and panicle length among the DJ, HM and HT lines (n = 4). (B) Comparison of internode lengths among the DJ, HM and HT lines (n = 4). (C) Comparison of grain filling among the DJ, HM and HT lines (n = 4). (D) Comparison of average weight of 30 seeds among the DJ, HM and HT lines (n = 4). (E) Panicle phenotype of DJ. (F) HM. (G) HT lines. Scale bar is 4 cm. Data are mean ± standard deviation (SD). Asterisk indicates significant differences compared with wild type (* p < 0.05 by Student’s t-test).

Figure 3

Generation of transgenic rice plants by over-expressing entire Osfuct gene in the HM mutant to confirm complementation. (A) Diagrammatic representation of the pCAMBIA3300 construct used for Osfuct transformation. RB, right border; FucT, entire Osfuct gene including the 2852-bp upstream sequence, and the 8066-bp downstream sequence; pVSsta and pVS1rep, neomycin phosphotransferase gene; pBR322 bom and ori; Kan, kanamycin resistance gene; Bar, phosphinothricin resistance gene; LB T35S, terminator of the 35S gene; _PFucT, Osfuct promoter, LB, left border sequence. (B) Morphological phenotype of restored lines (R3, R12, and R32) compared with the DJ, HM and HT lines. Scale bar is 17 cm. (C) Genotyping of restored lines (R3, R12, and R32) compared with the DJ, HM and HT lines. Gene specific (P1, P2, and P3) and T-DNA border sequence primer (P4) used for genotyping. The upper lane PCR products were amplified using gene-specific primers P1 and P3 (1.2 kb), while the lower lane PCR products were amplified using the P3 gene-specific primer and T-DNA border sequence primer P4 (0.6 kb). (D) Expression analysis of the Osfuct transcript in the rescued lines (R3, R12, and R32) compared with the DJ, HM and HT lines using RT–PCR. The expression level of actin1 was used as a loading control and gene specific primers (P5 and P6) were used for the RT–PCR analysis. (E–L) Comparison of anther phenotypes among the DJ, HM, HT and restored lines (R12), respectively. (M–P) Comparison of pistil phenotypes among the DJ, HM, HT and restored lines (R12), respectively. Scale bar is 1 mm.

Figure 4

Osfuct mutant affects pollen morphology, viability, and total number in the mutant (HM) and HT plant compared to Dongjin. Pollen grains were stained with 80% (w/v) potassium iodide and 10% iodine. The black stain indicates viable pollen grains, whereas non-viable pollen grains are stained yellow (A–D). In inset (black box), solid blue arrow indicates the non-viable pollen (A) Dongjin, (B) HM, (C) HT, and (D) rescue (R12). Scale bar is 10 µm. Pollen morphology of mutant, rescue, and wild-type plants (E–H): (E) Dongjin, (F) HM, (G) HT, and (H) rescue (R12). The mutant produced dramatically decreased pollen grains per anther. Pollen viability test showed a reduction in total pollen number and viability in the homozygous lines compared with the wild-type, reflecting the ill-developed anther. (I–K) Measurement of pollen area (µm) in mutant (HM) and HT plant, rescue (R12), and wild type (DJ) by scanning electron microscopy (SEM). Error bars show standard deviation. Scale bar is 10 µm. Data are mean ± SD (n ≥ 6). Asterisk indicates significant differences compared with wild type (* p < 0.05 by Student’s t-test).

Figure 5

Osfuct mutant sensitivity to salt stress. Effect of salt stress on growth of rice seedlings. Seedlings of the Dongjin (DJ) and mutant lines (HM) were exposed to a nutrient solution containing 0–200 mM NaCl. After 3 weeks, individual leaves were separated and shoot length (A) and weight were measured (B). Data are mean ± SD (n = 15). Asterisk indicates a significant difference compared with wild type (* p < 0.05 by t-test). Error bars show standard deviation.

Figure 6

Quantitative RT-PCR (qRT-PCR) results for the differentially expressed transcripts in wild type (DJ) and the Osfuct mutant (HM) to verify transcriptome profile data produced by microarray analysis. Data are mean ± SD (n = 3).

Figure 7

Possible function of Osfuct gene in growth, anther, and pollen development in rice. All up and downregulated genes together may affect growth and development in rice. Upregulated genes are in the red box and downregulated genes are in the green box. LecRLK, lectin receptor-like kinase; LRR-RLK, leucine-rich repeat receptor-like kinase; bHLH, basic/helix-loop-helix; DOF, DNA-binding one zinc finger; nsLTPs, non-specific lipid-transfer proteins; RGA4, resistance; EDE1, ENDOSPERM DEFECTIVE 1; KIF19, kinesin-like protein 19.