Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize

Abstract

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.

Keywords: Zea mays; embryo; fertilization; gynogenesis; haploid; phospholipase.

Figure 1. Fine mapping and structure of the ggi1/qhir1 locus

1. Key markers used during fine mapping. The scale refers to the reference sequence of chromosome 1 (RefGen_v3).

2. Segregation distortion at the ggi1/qhir1 for 16 markers (blue losanges) expressed as number of PK6 alleles divided by number of DH99 alleles and positioned on the same interval as described in (A).

3. GEvo analysis (https://genomevolution.org/coge/GEvo.pl) of the B73 and PK6 sequences. Regions with more than 95% similarity are marked by hatched boxes and linked by dotted lines. Gene models predicted in the B73 sequence are labeled with their GRMZM identifiers. Brown dashed lines delimit a 166‐kb region of B73 and a 80‐kb region of PK6. Both regions have only 10 kb in common and arrows indicate the 156‐kb region of B73 replaced in PK6 by a non‐related 70‐kb region. BAC clones used to establish the PK6 sequence are shown as yellow boxes below the PK6 sequence.

4. Schematic drawing of key recombinants using the B73 sequence as reference. Genome regions stemming from PK6 and DH99 are colored in red and blue, respectively. A color transition was chosen if the distance between the discriminating markers was more than 1 kb. The percentage of haploid induction of the corresponding plants is indicated as % (number of offspring analyzed) and classed as ind (inducer) or wt (wild‐type = non‐inducer).

5. Structure of predicted NLD proteins. Both wild‐type and NLD‐PK6 contain a patatin/phospholipase domain represented in green. The light gray box represents the phosphate/anion‐binding elements, the yellow horizontally hatched box the esterase box, and the dark box the catalytic dyad‐containing motif. Due to a frameshift, NLD‐PK6 lacks the 49 C‐terminal amino acid residues in comparison with wild‐type protein, which are replaced by 20 non‐related amino acid residues (positions 380–399 represented by red hatched box) ending with a premature STOP codon.

Figure 2. Pollen‐specific expression of NLD and temporal analysis of NLD promoter activity

1. A, B

   Relative NLD expression levels in major tissues of wild‐type line A188 (A) and in selected tissues of wild‐type line DH99 and inducer line PK6 (B) were determined by qRT–PCR. Values are means of at least three biological replicates ± SE. Asterisks (*) denote significant differences (P‐value < 0.05; t‐test).

2. C–F

   Temporal analysis of NLD promoter activity at different stages of pollen development visualized by histochemical detection of the GUS reporter. (C) In mature anthers of hemizygous plants, blue GUS staining was found in about 50% of the pollen grains, whereas no GUS staining was observed in the anther. (D) Determination of pollen stages by visualization of nuclei by DAPI (4′,6‐diamidino‐2‐phenylindole) fluorescence. (E) Bright field of the same pictures as in (D), showing GUS straining only in pollen grain at the tricellular stage when the sperm cells (white arrow) and vegetative cell (white arrow head) are present. (F) Observations of wild‐type silk at 17 h after pollination (HAP), and wild‐type ovules at 17 HAP, 48 HAP, and 72 HAP of plants crossed with transgenic pNLD::GUS pollen, revealed GUS activity in the pollen tube (black arrows) and in the embryo sac (arrow head) at 17 HAP, whereas no GUS staining was observed at 72 HAP. Scale bars, 50 μm (D, E) or 100 μm (C, F).

Figure 3. Subcellular localization of wild‐type and truncated NLD protein

1. A–D

   Confocal imaging of Arabidopsis root tips expressing either wild‐type NLD (A, B) or truncated NLD‐PK6 (C, D) fused to citrine fluorescent protein. Panels (B) and (D) are zooms of the red frames represented in (A) and (C), respectively. Signal of both protein fusions can be seen in the cytoplasm and endomembrane compartments, whereas only wild‐type NLD::citrine accumulated in the plasma membrane.

2. E–H

   Confocal imaging of pollen grain (E, F) or pollen tube (G, H) expressing either wild‐type NLD (E, G) or truncated NLD‐PK6 (F, H) fused to citrine fluorescent protein, under the control of the NLD promoter. Scale bars, 50 μm. Bright field merged to fluorescence channels are represented on the left, whereas fluorescence channel is represented on the right.

3. I

   Structured illumination microscopy (SIM) image (single image) of the male germ unit showing plasma membrane localization of wild‐type NLD::citrine protein in sperm cells (arrow); scale bar, 10 μm.