A Maize Male Gametophyte-Specific Gene Encodes ZmLARP6c1, a Potential RNA-Binding Protein Required for Competitive Pollen Tube Growth

Abstract

Members of the La-related protein family (LARPs) contain a conserved La module, which has been associated with RNA-binding activity. Expression of the maize gene GRMZM2G323499/Zm00001d018613, a member of the LARP family, is highly specific to pollen, based on both transcriptomic and proteomic assays. This suggests a pollen-specific RNA regulatory function for the protein, designated ZmLARP6c1 based on sequence similarity to the LARP6 subfamily in Arabidopsis. To test this hypothesis, a Ds-GFP transposable element insertion in the ZmLarp6c1 gene (tdsgR82C05) was obtained from the Dooner/Du mutant collection. Sequencing confirmed that the Ds-GFP insertion is in an exon, and thus likely interferes with ZmLARP6c1 function. Tracking inheritance of the insertion via its endosperm-expressed GFP indicated that the mutation was associated with reduced transmission from a heterozygous plant when crossed as a male (ranging from 0.5 to 26.5% transmission), but not as a female. Furthermore, this transmission defect was significantly alleviated when less pollen was applied to the silk, reducing competition between mutant and wild-type pollen. Pollen grain diameter measurements and nuclei counts showed no significant differences between wild-type and mutant pollen. However, in vitro, mutant pollen tubes were significantly shorter than those from sibling wild-type plants, and also displayed altered germination dynamics. These results are consistent with the idea that ZmLARP6c1 provides an important regulatory function during the highly competitive progamic phase of male gametophyte development following arrival of the pollen grain on the silk. The conditional, competitive nature of the Zmlarp6c1::Ds male sterility phenotype (i.e., reduced ability to produce progeny seed) points toward new possibilities for genetic control of parentage in crop production.

Keywords: RNA-binding protein; ZmLARP6c1; maize; pollen competition; pollen germination; pollen tube growth.

FIGURE 1

Bioinformatic analysis and expression profiling of the maize LARP6 family members points toward a role for ZmLarp6c1 in maize pollen. (A) Predicted conserved domain architecture of ZmLARP6c1. (B) Alignments of maize LARP6 amino acid sequences alongside consensus sequences for the LSA motif and the PAM2 motif. Gray shade, matched residues. (C) Phylogenetic tree, using the entire amino acid alignment and the distance method. The bar indicates a mean distance of 0.1 changes per amino acid residue. (D) Relative transcript levels of ZmLARP6 genes in mature pollen of maize. Total RNA was extracted from mature pollen for qRT-PCR. All data are means of three biological replicates with error bars indicating SD.

FIGURE 2

A Ds-GFP transposable element insertion line provides a tool to investigate Zmlarp6c1 function. (A) Sequencing confirmed that the insertion was in the 5th exon; thus, the mutation, likely a knockout, could be easily tracked genetically by observing GFP fluorescence in the endosperm. (B) PCR genotyping of progeny (using primers F1, R1, and Ac5) from a self-pollinated heterozygote confirms co-segregation of the insertion with the GFP marker, and helps clarify genotype (homozygote vs. heterozygote).

FIGURE 3

Maize ear projections demonstrate a strong male-specific transmission defect associated with the GFP-expressing Zmlarp6c1::DsgR82C05 allele. (A) Zmlarp6c1::Ds/+ crossed as a male (52 GFP kernels/420 total kernels = 12.4% transmission). (B) The same Zmlarp6c1::Ds/+ plant crossed as a female (246 GFP kernels/479 total kernels = 51.4% transmission). (C) Representative ear resulting from a cross of a homozygous Zmlarp6c1::Ds plant as a male to a wild-type female (average seed set = 319, standard deviation 101, n = 5 ears). (D) Representative ear resulting from a cross of a homozygous wild-type plant (a sibling from a closely related Zmlarp6c1::Ds family) as a male to a wild-type female (average seed set = 277, standard deviation 50, n = 5 ears).

FIGURE 4

Ds excision alleles derived from Zmlarp6c1::DsgR82C05 validate that the male specific transmission defect is due to loss of ZmLARP6c1 function. (A) PCR genotyping using primers F2 and R2 (Figure 2) documents isolation of two derivative alleles (dX550A8 and dX549B2) due to excision of the Ds-GFP element from Zmlarp6c1::DsgR82C05. W22: comparator wild-type inbred; MK: DNA size marker. (B) Sequencing of the derivative alleles shows that dX550A8 and dX549B2 are associated with an 8-bp and 6-bp footprints, respectively, in the Zmlarp6c1 coding sequence. (C) Zmlarp6c1-dX550A8 is associated with a male-specific transmission defect, with 6 out of 48 progeny from a heterozygous male outcross inheriting the footprint (12.5%). (D) Zmlarp6c1-dX549B2 segregates at a Mendelian ratio when transmitted through the male (13:11 from a heterozygous outcross).

FIGURE 5

Transmission of Zmlarp6c1::DsgR82C05 mutation is sensitive to pollen load. Outcomes of each separate ear with more than 10 kernels in the experiments from Table 2 are plotted. Pollination with heavy, near-saturating (HVY) pollen is associated with reduced transmission of Zmlarp6c1::Ds, relative to sparse (SPS) pollen (t-test, p = 0.002601). Moreover, linear regression (dotted line) indicates a significant correlation between total kernels per ear and transmission rate (p = 0.0003, R² = 0.647). Note that the two ears with ∼60% transmission are not significantly higher than 1:1, given the smaller populations they represent.

FIGURE 6

In vitro, pollen from Zmlarp6c1::Ds plants is associated with shorter pollen tubes and altered germination dynamics. (A) Representative fields of pollen germinated in vitro, at 15 min post-exposure to liquid media. Pollen was collected from either a homozygous Zmlarp6c1::Ds plant, or a comparator wild-type (WT) plant. Fields were scored blindly (four biological replicates of three plants of each genotype, at least 125 pollen grains from each plant), categorizing pollen grains as germinated (g), ruptured (r), or ungerminated (u). (B) Combined data from four biological replicates, at 15 and 30 min after plating in PGM. Modeling the categorical response using a multinomial baseline-category logit model indicates genotype is a significant predictor (p = 0.0047), as is timepoint (p = 1.02e-05). (C) Mutant pollen tube lengths at 30 min were significantly shorter than wild-type in all replications (Welch’s t-test, all p-values < 10^–15). Averaged across all four replicates, mutant pollen tubes were 31.5% shorter than wild-type sibling tubes.