A group-1 grass pollen allergen influences the outcome of pollen competition in maize

Abstract

Worldwide, 400 million people suffer from hay fever and seasonal asthma. The major causative agents of these allergies are pollen specific proteins called the group-1 grass pollen allergens. Although details of their antigenicity have been studied for 40 years with an eye towards immunotherapy, their function in the plant has drawn scant attention. Zea m 1 constitutes a class of abundant grass pollen allergens coded for by several genes that loosen the walls of grass cells, including the maize stigma and style. We have examined the impact of a transposon insertion into one of these genes (EXPB1, the most abundant isoform of Zea m 1) on the production of Zea m 1 protein, pollen viability, and pollen tube growth, both in vitro and in vivo. We also examined the effect of the insertional mutation on the competitive ability of the pollen by experimentally varying the sizes of the pollen load deposited onto stigmas using pollen from heterozygous plants and then screening the progeny for the presence of the transposon using PCR. We found that the insertional mutation reduced the levels of Zea m 1 in maize pollen, but had no effect on pollen viability, in vitro pollen tube growth or the proportion of progeny sired when small pollen loads are deposited onto stigmas. However, when large pollen loads are deposited onto the stigmas, the transposon mutation is vastly underrepresented in the progeny, indicating that this major pollen allergen has a large effect on pollen tube growth rates in vivo, and plays an important role in determining the outcome of the pollen-pollen competition for access to the ovules. We propose that the extraordinary abundance (4% of the extractable protein in maize pollen) of this major pollen allergen is the result of selection for a trait that functions primarily in providing differential access to ovules.

Figure 1

Mu insertion into EXPB1 and its effect on Zea m 1 content of pollen. (a) Cartoon showing the structure of EXPB1 and location of the Mu insertion (exons denoted with boxes). Also indicated are the locations of primers used for PCR screening. (b) Mu is inserted near the intron border flanking the fourth exon. (c) Portion of a 2-D gel image of wild type (EXPB1) pollen protein showing the Zea m 1 isoforms, which were identified by immunoblotting. (d) Relative amount of total Zea m 1 protein extracted from pollen of EXPB1/EXPB1 and expb1/expb1 plants. (Mean±SE; N = 2; t = 9.15; p = 0.035).

Figure 2

Pollen viability and pollen performance in vitro and in vivo. (a) Percentage of viable pollen, based on staining with thiazolyl blue (mean±SE, N = 20–22 plants). (b) Micrograph of pollen stained with thiazolyl blue. Viable pollen stained dark purple. (c) Pollen tube growth in vitro (mean±SE, N = 20–22). Bars with different letters of the alphabet differ significantly using Tukey pairwise comparisons with the overall probability adjusted for multiple comparisons.

Figure 3

Transmission rate of expb1 as a function of the size of the pollen load from EXPB1/expb1 plants (mean±SE, N = 4).

Figure 4

Pollen tube from EXPB1 pollen growing through ovary tissue for 22 h after pollination. Ovaries were stained with 0.1% aniline blue for 30 min and then examined under a fluorescence microscope.