Washington State University

Pollen tube growth in the white pollen mutant

--Karen Hansen and Loverine P. Taylor

We are investigating the physiological and molecular basis for the self-sterility associated with flavonoid deficiency in white pollen plants. This work represents a continuation and expansion of investigations initiated by E. H. Coe, EH and co-workers and we gratefully acknowledge his encouragement and generosity. The white pollen mutant is associated with stable recessive alleles at the duplicate loci, C2 and Whp (Coe, EH et al., J. Hered. 72:318, 1981). Both of these genes encode chalcone synthase (Franken et al., EMBO J. 10:2605, 1991), the enzyme that catalyzes the first step in flavonoid biosynthesis. In the absence of chalcone synthase (CHS) activity, plants lack flavonoids in all tissues including pollen (Coe, EH et al., 1981; D. Styles, ED, unpublished data; L. P. Taylor, LP and C. W. Nagel, CW, in preparation).

Characterization of a similar phenomenon in petunia shows that white pollen is unable to germinate either in vivo or in vitro (Taylor and Jorgensen, J. Hered. 83, 1992). To determine if the maize white pollen mutant (c2/c2 whp/whp) exhibits a similar pollen phenotype, we compared in vitro germinated white pollen with an isogenic line differing by a single functional copy of CHS (C2/c2 whp/whp). Pollen from both the mutant and functional lines readily germinates on semi-solid media and produces tubes that are indistinguishable even after 8 hours of growth. However, in vitro pollen tube growth rates never approach the rates seen in vivo, suggesting that differences between the mutant white pollen and functional pollen may only be apparent in vivo.

Self-crosses of the white pollen mutant produce virtually no seed but the plants are female fertile and crosses of C2/c2 whp/whp pollen onto c2/c2 whp/whp silks produce fully filled ears. We confirmed an early suggestion of S. A. Modena, SA (MNL 56:47, 1982) that if the white pollen silks are shortened, the mutant pollen tubes grow long enough to achieve fertilization. The ear pictured in Figure 1 is a white pollen self-cross. A window was cut through the husks, the silks within the window area were carefully extracted, cut to a uniform 1.5cm length, and self pollinated. Nearly full kernel set occurred in the window area.

Figure 1. White pollen self-cross 45 days post pollination.

This experiment indicates that white pollen tubes can grow at least 1.5cm. To determine where pollen tube growth ceases, we examined self-crossed white pollen silks throughout their length for the presence of pollen tubes. White pollen silks were harvested 48 hrs. after pollination with either yellow C2/c2 whp/whp pollen (control) or white c2/c2 whp/whp pollen, fixed in acetic acid/formaldehyde/ethanol (1:1:8), cleared in 8N NaOH, and stained with decolorized aniline blue (0.01% in M/15 K2HPO4). Pollen tubes are visualized within the silks by fluorescence microscopy of the brightly stained callose deposits that are characteristic of growing pollen tubes.

Pollen grains caught by the silk hairs hydrate then germinate. The growing tube penetrates and moves into the body of the silk where it grows parallel to the vascular bundles. On a representative group of silks (17) pollen tube length was scored relative to the total silk length (Figure 2). Of the control pollinations, >98% of the germinated C2/c2 whp/whp pollen produced a tube at least 2mm in length. The dramatic decrease in the number of tubes approximately 1/3 of the distance down the silk suggests that some selective mechanism may be operating in this region. The pollen tube number stays constant throughout the middle one-third of the silk and then drops to 9% of the germinated grains in the portion of the silk closest to the embryo sac. Most silks examined had 1-3 tubes at the base where it was detached from the kernel. We did not examine the micropylar region.

Figure 2. Pollen tube growth relative to silk length of C2/c2 whp/whp (n) and c2/c2 whp/whp (l).

In sharp contrast to the pattern of functional pollen tube growth, only 24% of the germinated white pollen grains produce a tube more than 2mm in length. The number of growing tubes rapidly drops off and no tubes are observed in the lower two-thirds of the silk. The longest white pollen tube length recorded was 5cm which represents 37% of the total silk length. The premature termination of white pollen tubes is easily observed because the tube begins to grow in a meandering pattern and accumulates greater than normal amounts of callose 1-4mm from the tip. This undulating pattern and callose deposition was never observed in the control pollinations. The white pollen grains and tubes showed no gross abnormalities in germination and growth until this point, which agrees with our in vitro germination observations. These results suggest that unlike petunia, flavonoids may not be essential for pollen germination in maize, however, they are required for the sustained tube growth necessary to achieve fertilization.

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