Sporophytic control of pollen tube growth and guidance in maize

Abstract

Pollen tube germination, growth, and guidance (progamic phase) culminating in sperm discharge is a multi-stage process including complex interactions between the male gametophyte as well as sporophytic tissues and the female gametophyte (embryo sac), respectively. Inter- and intra-specific crossing barriers in maize and Tripsacum have been studied and a precise description of progamic pollen tube development in maize is reported here. It was found that pollen germination and initial tube growth are rather unspecific, but an early, first crossing barrier was detected before arrival at the transmitting tract. Pollination of maize silks with Tripsacum pollen and incompatible pollination of Ga1s/Ga1s-maize silks with ga1-maize pollen revealed another two incompatibility barriers, namely transmitting tract mistargeting and insufficient growth support. Attraction and growth support by the transmitting tract seem to play key roles for progamic pollen tube growth. After leaving transmitting tracts, pollen tubes have to navigate across the ovule in the ovular cavity. Pollination of an embryo sac-less maize RNAi-line allowed the role of the female gametophyte for pollen tube guidance to be determined in maize. It was found that female gametophyte controlled guidance is restricted to a small region around the micropyle, approximately 50-100 microm in diameter. This area is comparable to the area of influence of previously described ZmEA1-based short-range female gametophyte signalling. In conclusion, the progamic phase is almost completely under sporophytic control in maize.

Fig. 1.

Germination rates of pollen from various monocot and dicot species on silks of maize and Tripsacum dactyloides, respectively. Silks of maize (A–D) and Tripsacum dactyloides (E–H) were pollinated with pollen from different plant species and incubated in a humid chamber at room temperature. All specimen were stained with aniline blue and false red colour animated for better contrast. Germination of maize pollen grains is shown in (A, B) and (G), that of T. dactyloides pollen in (C) and (E, F), while Arabidopsis thaliana pollen are shown in (D) and (H). To determine the relative pollen germination efficiency (I, J), germination was evaluated after 3–7 h without staining and each value related to the germination rate after self-pollination. See Supplementary Table S1 at JXB online for details. Abbreviations: Zm, Zea mays; Td, Tripsacum dactyloides; Pn, Poa nemoralis; Lm, Lolium multiflorum; Os, Oryza sativa; At, Arabidopsis thaliana; Ll, Lilium longiflorum. Scale bars: 200 μm.

Fig. 2.

Pollen tube growth range in silks of maize inbreed line A188. (A) Cob segments have been placed in a humid chamber and were pollinated with pollen from maize as well as tetraploid and hexaploid Tripsacum dactyloides, respectively. 16 h after incubation at room temperature, silks were cut in 2 cm pieces with proximal ends varying in length. Specimens were fixed, analysed after aniline blue staining and false red colour animated for better contrast (B–G). (B, C) Maize pollen tubes outside and inside the silk tissue (arrow in C) within the transmitting tract (bracket). (D, E) Pollen tubes of both species, maize and T. dactyloides grow towards the transmitting tracts surrounding the parenchymal cells (arrow in E). (F) Some T. dactyloides pollen tubes grew outside the transmitting tract (arrows) and (G) arrested after shorter growth than those inside the transmitting tracts (brackets; pollen tubes inside the TT are marked with arrowheads). T. dactyloides pollen tube (arrow) growth arrest was observed in silk segments at 2–8 cm distance from the area of pollination. Pollen tubes inside transmitting tracts (brackets; pollen tubes marked with arrowheads) continued growth. (H) Silk segments containing pollen tubes were counted and set in relation to the total number of silks investigated. Brackets indicate transmitting tracts. Bold arrows indicate pollen tube growth direction towards the ovary. Scale bars: 200 μm.

Fig. 3.

Pollen tubes growth range in silks of maize inbred line K55 harbouring different allele combinations of Ga1s. Cob segments were placed in a humid chamber and were pollinated as indicated. After 16 h incubation at room temperature, silks were cut, stained and pollen tubes counted as described before. (A) Silks dominant for Ga1s. (B) Silks heterozygous for Ga1s. (C) Silks recessive for Ga1s (ga1/ga1).

Fig. 4.

Cross-sections of maize and T. dactyloides silks stained with aniline blue. (A) In an unpollinated maize silk, xylem elements (brackets) of the two vascular bundles show strong aniline blue staining whereas other cell walls lead to a light background signal. The transmitting tract (encircled) is composed of small longitudinal cells in close proximity to the xylem elements. (B) 24 h after pollination with wild-type maize pollen, pollen tubes appear as large, round, and brightly stained structures growing in the intercellular space between TT cells (only one vascular bundle is visible). (C) After pollination of Ga1s/Ga1s silks with recessive pollen (ga1), some pollen tubes (pt) grew outside of the TT towards the ovule. (D) The same phenomenon can be seen regularly in maize silks pollinated with T. dactyloides pollen. If pollinated silks are cut at the site of pollination, several pollen tubes grew outside the TT right below the epidermis (four pollen tubes between parechymal cells are visible in the onset). (E) These pollen tubes stop their growth earlier and are therefore less abundant in more distal parts of the silk. (F) Compared to maize, T. dactyloides transmitting tracts are more deeply embedded into the silk tissue and contain only one vascular bundle (bracket) with associated TT (encircled). Cross-section of pollinated silks show pollen tube growth exclusively in TT in T. dactyloides silks pollinated either with T. dactyloides (G) or maize (H) pollen. Scale bars: 50 μm.

Fig. 5.

Pollen tube growth and attraction in the micropylar region of the ovule. (A) CLSM longitudinal section through a WT ovule displaying a mature female gametophyte (FG). (B) fg-RNAi mutant ovule lacking a functional FG that is completely disintegrated after stage FG5 (Srilunchang et al., 2010). (C) WT ovule 24 h after pollination and aniline blue staining (false colour red staining was used for better visibility of pollen tubes). Several pollen tubes arrived at the ovule surface and grew towards the micropylar region. One pollen tube grew inside the micropyle and released its contents inside the receptive synergid. The female gametophyte is encircled. The inset indicates the enlarged region shown in (D). (D) Only one pollen tube succeeded in entering the micropyle (circle) and additional pollen tubes seem to be no longer attracted by the micropylar region. (E, F) Ovules of the fg-RNAi mutant line: the female gametophyte is disintegrated (encircled) and pollen tubes (arrowheads) grew in 50–100 μm proximity to the centre of the micropylar cone (circle), but did not enter. Abbreviations: ap, antipodal cells; cc, central cell; des, degenerated embryo sac; ec, egg cell; es, embryo sac; mp, micropyle; pn, polar nuclei. Scale bars: 50 μm.

Fig. 6.

Summary of comparative progamic pollen germination and growth in maize and Arabidopsis. Although anatomical structures are named differently, Phase I–III can be widely homologized between maize and Arabidopsis. (A) Scheme showing progamic pollen tube growth failures observed in this survey. Early growth arrest was observed with pollen of rice, Poa, Lolium, and Arabidopsis. Lily pollen did not germinate or stopped growth before leaving the silk hair. (B) Pollen tubes of these species arrested in Phase I (lily) or Phase II (most grasses and Arabidopsis). Tripsacum and incompatible maize pollen tubes show two linked phenomena. Many pollen tubes are mistargeted and grow outside the transmitting tract. Those pollen tubes which find their way into the transmitting tract stop growth after a few centimetres (Phase III). After passing the abscission zone in maize, pollen tubes reach the end of the transmitting tract and enter the ovarial cavity. Growth in the ovarial cavity of grasses and, accordingly, growth on the septum surface, funiculus, and ovule surface of Arabidopsis represent Phase IV. Whereas female gametophytes of Arabidopsis contribute to funicular and micropylar guidance, maize embryo sacs only provide micropylar guidance cues (Phase V). As a sporophytic default status, maize pollen tubes are guided towards the micropyle, but do not enter an area about 100 μm in diameter around the micropylar cone. Ovules are shown in green, transmitting tracts in grey, embryo sacs in red, and male gametophytes in blue. Abbreviations: az, abscission zone; es, embryo sac; fu, funiculus; o, ovule; p, pollen; pl, placenta; pt, pollen tube; sh, silk hair; st, stigma; tt, transmitting tract.