MYB97, MYB101 and MYB120 function as male factors that control pollen tube-synergid interaction in Arabidopsis thaliana fertilization

Abstract

Pollen tube reception involves a pollen tube-synergid interaction that controls the discharge of sperm cells into the embryo sac during plant fertilization. Despite its importance in the sexual reproduction of plants, little is known about the role of gene regulation in this process. We report here that the pollen-expressed transcription factors MYB97, MYB101 and MYB120 probably control genes whose encoded proteins play important roles in Arabidopsis thaliana pollen tube reception. They share a high amino acid sequence identity and are expressed mainly in mature pollen grains and pollen tubes. None of the single or double mutants of these three genes exhibited any visible defective phenotype. Although the myb97 myb101 myb120 triple mutant was not defective in pollen development, pollen germination, pollen tube growth or tube guidance, the pollen tubes of the triple mutants exhibited uncontrolled growth and failed to discharge their sperm cells after entering the embryo sac. In addition, the myb97 myb101 myb120 triple mutation significantly affected the expression of a group of pollen-expressed genes in mature pollen grains. All these results indicate that MYB97, MYB101 and MYB120 participate in pollen tube reception, possibly by controlling the expression of downstream genes.

Figure 1. Identification of the MYB genes.

(A) Phylogenetic analysis of the MYB proteins. (B) Expression patterns of the MYB genes, as revealed by RT-PCR. (C) Expression of the MYB genes in mature pollen grains, as revealed by qRT-PCR. gDNA, genomic DNA; Rt, roots; St, stems; Lf, leaves; If, inflorescences; Mp, mature pollen grains; Sq, siliques and Sl, seedlings.

Figure 2. MYB97, MYB101 and MYB120 were expressed in pollen grains and pollen tubes.

(A) to (D) Expression pattern of MYB97, as shown by GUS staining in an inflorescence (A), flower (B), mature pollen grains (C) and pollen tube (D). (E) to (H) Expression pattern of MYB120, as shown by GUS staining in an inflorescence (E), flower (F), mature pollen grains (G) and pollen tube (H). (I) to (L) Expression pattern of MYB101, as shown by GUS staining in an inflorescence (I), flower (J), mature pollen grains (K) and pollen tube (L). Bars = 2 mm in (A), (E) and (I); 1 mm in (B), (F) and (J) and 40 µm in (C), (D), (G), (H), (K), (L).

Figure 3. Molecular characterization of myb97, myb101 and myb120 mutants.

(A) Schematic diagrams of the MYB gene structures and T-DNA insertion sites in the mutants. The gray and white boxes indicate the translated and untranslated regions, respectively. (B) The expression levels of MYB97, MYB101 and MYB120 genes in wild type (WT) and the triple homozygous mutants, as revealed by qRT-PCR.

Figure 4. Complementation of the myb97-1 myb101-1 myb120-3 mutant.

The fertility of the myb97-1 myb101-1 myb120-3 triple mutant was restored completely by transformation with MYB97, MYB101, MYB120, pMYB101::MYB33 and pMYB101::MYB81 complementation constructs. The red arrow indicates the unfertilized ovules. Bars = 2 mm.

Figure 5. The myb97 myb101 myb120 triple mutations did not affect the formation of pollen grains.

The pollen grains were examined by Alexander staining, DAPI staining and scanning electron microscopy (SEM). (A) to (C) WT pollen grains. (D) to (F) myb97-1 myb101-1 myb120-3 pollen grains. (G) to (I) myb97-1 myb101-2 myb120-3 pollen grains. (J) to (L) myb97-1 myb101-3 myb120-3 pollen grains. Bars = 40 µm in (A), (D), (G) and (J); 20 µm in (B), (E), (H) and (K); and 30 µm in (C), (F), (I) and (L).

Figure 6. The myb97 myb101 myb120 triple mutant pollen tubes did not stop growing and failed to discharge sperm cells into the embryo sacs.

(A) to (D) The reciprocal crosses between myb97-1 myb101-1 myb120-3 and wild type. (A) A Col silique pollinated with Col pollen. (B) A Col silique pollinated with myb97-1 myb101-1 myb120-3 pollen. The white arrow indicates the overgrowing pollen tubes. (C) A myb97-1 myb101-1 myb120-3 triple mutant silique pollinated with Col pollen. (D) A myb97-1 myb101-1 myb120-3 triple mutant silique pollinated with myb97-1 myb101-1 myb120-3 pollen. The white arrow indicates the overgrowing pollen tubes. (E) to (H) The reciprocal crosses between myb97-1 myb101-2 myb120-3 and wild type. (E) A Col silique pollinated with Col pollen. (F) A Col silique pollinated with myb97-1 myb101-2 myb120-3 pollen. The white arrow indicates overgrowing pollen tubes. (G) A myb97-1 myb101-2 myb120-3 triple mutant silique pollinated with Col pollen. (H) myb97-1 myb101-2 myb120-3 triple mutant silique pollinated with myb97-1 myb101-2 myb120-3 pollen. The white arrow indicates overgrowing pollen tubes. (I) to (L) The reciprocal crosses between myb97-1 myb101-3 myb120-3 and wild type. (I) A Col silique pollinated with Col pollen. (J) A Col silique pollinated with myb97-1 myb101-3 myb120-3 pollen. The white arrow indicates overgrowing pollen tubes. (K) A myb97-1 myb101-3 myb120-3 silique pollinated with Col pollen. (L) A myb97-1 myb101-3 myb120-3 silique pollinated with myb97-1 myb101-3 myb120-3 pollen. The white arrow indicates overgrowing pollen tubes. (M) to (P) Images showing the normal growth pattern of a wild type pollen tube (M) and the overgrowth patterns of myb97-1 myb101-1 myb120-3 (N), myb97-1 myb101-2 myb120-3 (O) and myb97-1 myb101-3 myb120-3 (P) pollen tubes, as indicated by white arrows. Bars = 100 µm in (A) to (L); 40 µm in (M) to (P).

Figure 7. Subcellular localization of GFP-MYB fusion proteins.

(A) to (C) Control, showing that the GFP signal from the p35S::GFP construct was distributed throughout the onion epidermal cell. (D) to (F) The GFP signal from the p35S::GFP-MYB97 construct localizes to the nucleus. (G) to (I) The GFP signal from the p35S::GFP-MYB120 construct localizes to the nucleus. (J) to (L) The GFP signal from the p35S::GFP-MYB101 construct localizes to the nucleus. Bars = 1 mm in (A) to (C), (G) to (I); 100 µm in (D) to (F) and (J) to (L).

Figure 8. Transactivational activity assays.

(A) Schematic diagrams of the different constructs used for transactivation activity assays. (B) The transactivation activity of MYB97 and MYB101 in yeast. DNA-BD, GAL4 DNA-binding domain; GAL4 AD, GAL4 activation domain; NLS, nuclear localization signal; MCS, multiple cloning site; MYB97, MYB101 and MYB120, the coding sequences (CDS) corresponding to MYB97, MYB101 and MYB120.

Figure 9. MYB101 binds to the MYBGAHV (TAACAAA) cis-element in the DG1, DG2 and DG3 promoters in vitro.

(A) to (B) The expression of DG1, DG2, and DG3 were significantly reduced in the myb97 myb101 myb120 triple mutant, as revealed by RT-PCR (A) and qRT-PCR (B). (C) The sequences of the oligonucleotides used in the EMSA experiments. DG1WT, DG2WT and DG3WT are the wild-type versions of the MYBGAHV (TAACAAA) cis-elements (underlined) in the DG1, DG2 and DG3 promoters, respectively. The TAACAAA motifs were mutated as indicated by lowercase letters in the mDG1WT, mDG2WT and mDG3WT sequences. (D) MYB101 is able to bind to the MYBGAHV cis-element (TAACAAA) in the DG1 promoter. Lanes 1 and 2 show reactions to which the MYB101 protein or the biotin-labeled DG1WT oligonucleotide was added, respectively. As shown in lane 3, a shift (black triangle) was observed when the MYB101 protein was added to the reaction containing the biotin-labeled DG1WT oligonucleotide. Lanes 4 to 7 show reactions in which unlabeled oligonucleotides were added to the binding reactions to compete with biotin-labeled DG1WT oligonucleotide. The competition becomes increasingly apparent with the unlabeled DG1WT oligonucleotide added at 50×, 250× and 500× molar excess in lanes 4, 5 and 6, respectively. Lane 7 shows that the unlabeled mDG1WT oligonucleotide competed only weakly, even at 500× molar excess. These results were reconfirmed in independent EMSA experiments. (E) MYB101 is able to bind to the MYBGAHV cis-element in the DG2 promoter. The binding reaction containing the MYB101 protein and the biotin-labeled DG2WT oligonucleotide causes a clear shift. The unlabeled DG2WT oligonucleotide competed fully at 500× molar excess. No competition was observed when the unlabeled mDG2WT oligonucleotide was used at 500× molar excess. These results were reconfirmed in independent EMSA experiments. (F) MYB101 is able to bind to the MYBGAHV cis-element in the DG3 promoter. The biotin-labeled DG3WT oligonucleotide was mixed with the MYB101 protein, and a shift was observed in the binding reaction. The unlabeled DG3WT oligonucleotide competed fully at 500× molar excess. Only weak competition was observed when unlabeled mDG3WT oligonucleotide was used at 500× molar excess. These results were reconfirmed in independent EMSA experiments.