MAA3 (MAGATAMA3) helicase gene is required for female gametophyte development and pollen tube guidance in Arabidopsis thaliana

Abstract

The female gametophyte plays a central role in the sexual reproduction of angiosperms. We previously isolated the maa3 (magatama3) mutant of Arabidopsis thaliana, defective in development of the female gametophyte, micropylar pollen tube guidance, and preventing the attraction of multiple pollen tubes. We here observed that the nucleolus of polar nuclei is small, and that the fusion of polar nuclei often did not occur at the time of pollination. The MAA3 gene encodes a homolog of yeast Sen1 helicase, required for RNA metabolism. It is suggested that MAA3 may regulate RNA molecules responsible for nucleolar organization and pollen tube guidance.

Fig. 1

maa3 and wild-type female gametophytes in maa3 heterozygote pistils. (A–E) FGs with the maa3 phenotype. (F–J) FGs with the wild-type phenotype. (A) maa3 FG in the early FG5 stage. (B) maa3 FG in the late FG5 stage. The nucleoli of polar nuclei are magnified four times in the inset. The two nucleoli were small, without internal structure, and their sizes were different. (C) FG in stage FG4. This was in the same pistil as wild-type late FG5 (G) and wild-type FG6 (H). (D, E) maa3 FG in the FG6 stage in two microscopic foci. (F) Wild-type late FG5. (G) Wild-type late FG5. Another synergid and an egg cell are out of focus. The nucleoli of polar nuclei were magnified four times in the inset to show the internal, round structure. (H) Wild-type FG6. (I, J) Two-nucleate endosperm stage of the wild type in two microscopic foci. (A) and (F) were in pistil 3 in Supplementary Table S1, (B) in pistil 4, (C), (G) and (H) in pistil 2, and (D), (E), (I) and (J) in pistil 5. Pistils 2 and 3 were fixed and observed before anthesis. Pistils 4 and 5 were after anthesis and autopollination. Scale bar = 20 μm. Arrowhead, nucleolus of the four-nucleate stage; a, nucleolus of the antipodal cell; c, nucleolus of fused polar nuclei in the central cell; e, nucleolus of the egg cell; en, nucleolus of the endosperm; nv, nucleolar vacuole; p, nucleolus of polar nuclei; s, nucleoli of the synergid cell; z, nucleolus of the zygote.

Fig. 2

Schematic drawing of the genomic structure of the MAA3 locus. The upper line shows position 60,001–70,000 bp of the ATFCA4 contig, and the cDNA structure is shown below. Green and blue boxes indicate coding and non-coding exon regions of the MAA3 gene, respectively. An arrow indicates the direction of transcription. Orange and white boxes indicate the neighboring genes (coding regions in orange) predicted by GENSCAN software. The genomic fragment between two points labeled C was used for complementation. The position of the T-DNA insertion and the inverse PCR product is shown.

Fig. 3

Expression analysis of the MAA3 gene. RT–PCR of the MAA3 gene with the ACT8 gene as control. Fl, flower; sl, silique; vm, shoot apex including vegetative meristem; lf, leaf; rt, root; g, genomic DNA.