Sperm cells are passive cargo of the pollen tube in plant fertilization

Abstract

Sperm cells of seed plants have lost their motility and are transported by the vegetative pollen tube cell for fertilization, but the extent to which they regulate their own transportation is a long-standing debate. Here we show that Arabidopsis lacking two bHLH transcription factors produces pollen without sperm cells. This abnormal pollen mostly behaves like the wild type and demonstrates that sperm cells are dispensable for normal pollen tube development.

Figure 1. The drop1− drop2− pollen grains lack sperm cells

a, Left: phylogenic tree shows the relationship of three DROP proteins. The conserved bHLH domain is indicated by a yellow box. Right: heatmap indicating the relative expression level of each DROP gene in different tissues. Values were taken from RNA-seq data using designated tissues. b–d, Alexander’s staining (b), scanning electron microscopy (c) and DAPI-staining (d) of drop1+/− drop2−/− pollen. Blue arrows indicate WT-looking pollen; yellow arrows indicate water drop-like pollen; red arrows indicate collapsed pollen. e, Statistical analysis of pollen defects in drop mutants as indicated. f–h, Bright-field-UV merged images of mature pollen grains of drop1−/− drop2+/− qrt1 mutant. i, Statistical analysis of pollen defects in drop1−/− drop2+/− qrt1. j,k, Transmission electron microscopy of WT and drop1− drop2− pollen, respectively. Blue arrow indicates abnormal cytoplasmic protrusion. l–q, Confocal laser scanning microscopy of WT and drop1− drop2− pollen expressing different reporters. The vegetative marker ProLAT52:GFP is present in both WT (green in l) and drop1− drop2− mutants (m), but the generative cell marker ProHTR10:HTR10-RFP (red in l) is absent from drop1− drop2− pollen (m). In n and o, expression of the double marker line ProLAT52:H2B-GFP (green, vegetative nucleus) ProDUO1:TdTOM (red, generative cell) in WT and drop1− drop2− pollen, respectively. In p and q, the vegetative marker ProWIP1:WIP1-TdTOM (red) is expressed in both WT and drop1− drop2− pollen, respectively. r, Quantification of expression levels of vegetative and sperm cell-specific genes in semi-in vivo grown WT (blue bars) and in drop1− drop2− (red bars) pollen tubes. PT, pollen tube; RPKM, reads per kilobase per million reads. Scale bars, 20 μm (b); 10 μm (c,d,f,g,h); 5 μm (j–q).

Figure 2. Sperm-less drop1− drop2− mutant pollen tubes grow normally toward the ovule

a–d, In vitro germination and growth of WT (a,b) and drop1− drop2− pollen (c,d). Panels a and c show bright-field microscopy images; b and d show epifluorescence microscopy of DAPI-stained pollen tubes. Yellow arrowhead points to the vegetative nucleus; red arrowhead indicates the sperm cell nucleus. e, Statistical analysis of in vitro germination ratio of WT and drop1− drop2− pollen. f,g, Aniline blue staining of emasculated WT pistil that was pollinated with WT pollen (f) and drop1− drop2− pollen (g) 4HAP, respectively. The orange line marks the furthest point of pollen tube growth. Note that pollen tubes of drop1− drop2−, similar to those of WT, reach the end of the pistil. h, Statistical analysis of the growth speed of in vivo pollen tubes in pistils 4 HAP and 6 HAP. i–n, SIV PT attraction assay tracing guided pollen tube growth towards recombinant attraction protein AtLURE1.2 in a gelatin bead (shaded with light green). Asterisk indicates the tip of a growing pollen tube at different points. The identity of nuclei in j,m was revealed by DAPI staining (k,n, respectively). Red arrowhead marks the sperm cell nucleus; yellow arrowhead indicates the vegetative nucleus. o–t, WT and drop1− drop2− pollen tube targeting (o for WT and q for the mutant) and rupture (p for WT and r for the mutant). In s, seed development is initiated from WT ovules (yellow arrowheads indicate endosperm nuclei, and green arrow head the embryo), but not after the rupture of drop1− drop2− pollen tubes. In t, the blue arrowhead indicates the central cell and the red arrowhead indicates the egg cell. In e and h, data and error bars represent the mean ± s.d. Scale bars, 20 μm (a,c,s,t); 10 μm (b,d); 200 μm (f,g); 100 μm (i–n); 50 μm (o–r).