Principles and Practices in Plant Ecology: Allelochemical Interactions (1999) p. 119-121

8 - Microspectrofluorimetry of Intact Secreting Cells, with Applications to the Study of Allelopathy

Victoria V. Roschina and Eugenia V. Melnikova Pollen-pistil interaction

The experiments on pollination included fluorescence measurements 2 to 5 min following germination and 1 month following ovary development. The fluorescence spectra of the Hippeastrum stigma was analyzed after pollination by foreign and own pollen (Figure 8.8). When pollen of wind-pollinated species such as Dactylis glomerata L. (Figure 8.8), Populus balsamifera L., and Betula verrucosa were added, the weak own intrinsic fluorescence of the Hippeastrum pistil responded to the pollen addition on the stigma. The character of spectra was changed by both cross-pollination and self-pollination, where new peaks arose (Figures 8.8, i-iii), whereas pollination by pollen from insect-pollinated Hemerocallis fulva L. only increased the whole influorescence intensity (Figure 8.10, ii). After 1 month of observation, the ovary formation was at variance with that following cross- or self-pollination. In other cases, fruit maturation was not seen.

Simultaneously with the measurements on the pistil, the fluorescence of the same species pollen added on the Hippeastrum stigma was also recorded (Figure 8.8). The pollen grains of foreign pollen weakly or practically did not change the autofluorescence spectra. However, own-pollen fluorescence intensity decreased 2 to 3 min after its addition on the pistil. Its maximum at 480 nm shifted to the long wavelength region (Figure 8.8). This phenomenon appeared to be associated with the chemosensory features of the surfaces of both the stigma and the pollen grains.

FIGURE 8.8 (A) The fluorescence spectra of stigma of Hippeastrum without (- - -) and with pollen grains (____). (i) + Dactylis glomerata pollen, (ii) + Hemerocallis fulva pollen, (iii) + own pollen (cross – pollination by pollen from other plants of the Hippeastrum (Amaryllidaceae), (iv) + own pollen (self-pollination by pollen of the same flower). (B) The fluorescence spectra of pollen (of I, ii, or iii) with (- - -) and without (____) Hippeastrum stigma interaction. (i) Dactylis glomerata, (ii) Hemerocallis fulva, (iii) Hippeastrum sp. (C) Outline of pollen showing the position of optical sound, S. Pollen-Pollen Interaction in Mixtures

One of the suitable modes of estimating pollen-pollen interactions is observation of fast changes in their autofluorescence. The responses are obviously dependent on both the composition of the excretion of donor pollen and the chemosensory peculiarities of the surface component of the plant-acceptor pollen. In this section we will consider the chemical interactions between dry pollen grains, for example, through a communication by volatile excretion, unlike our experiments with moistened pollen and leachates from pollen (Roschina and Melnikova, 1996).

The present data involve pollen of meadow wind-pollinated species; forest-living wind-pollinated species; meadow wind-pollinated and insect-pollenates species, grown in flower gardens; and weeds.

The fluorescence spectra of pollen mixtures may reflect initial fast responses in pollen allelopathy. One example is shown in Figure 8.13, where the fluorescence spectra of mixtures from wind-pollinated herbaceous meadow plants such as meadow foxtail (Alopecurus pratensis L.) and orchardgrass (Dactylis glomerata L., Poaceae), and cultural flower garden species poppy (Papaver orientale L., Papaveraceae) and day lily (Hemerocallis fulva L., Liliaceae) are given. The changes are mainly in the fluorescence intensity. The value is 1.5-fold higher in Alopecurus in a mixture of Alopecurus and Papaver (var. i, ii), whereas poppy has no visible shifts. In conrast, meadow foxglove decreases its autofluorescence intensity and shifts the main maximum to the long wavelength region in a mixture with day lily pollen (var. iii, iv). The latter shows no changes in its autofluorescence. When pollen grains of Hemerocallis and Dactylis (var. v, vi) are mixed, orchardgrass undergoes significant changes and a new maximum at 480 nm arises, with major maximum at 525 nm becomes more flat, and red fluorescence with maximum at 650 nm is more obvious, while the intensity of fluorescence in maxima decreases more than twofold. The autofluorescence of day lily pollen also decreases. If pollen of orchardgrass and poppy are mixed, the fluorescence of both remains unchanged (var. vii, viii).

FIGURE 8.13 The fluorescence spectra of pollen grains in mixtures of allelopathically active species. Pollen fluorescence spectra (___) without and (----) with the foreign pollen in mixture. (i) Pollen of Alopecurus pratensis without and with pollen of Papaver orientale; (ii) pollen of Papaver orientale without and with pollen of Alopecurus pratensis; (iii) pollen of Alopecurus pratensis without and with pollen of Hemerocallis fulva; (iv) pollen of Hemerocallis fulva without and with pollen of Alopecurus pratensis; (v) pollen of Dactylis glomerata without and with pollen of Hemerocallis fulva; (vii) pollen of Hemerocallis fulva without and with pollen of Dactyllis glomerata; (vii) pollen of Alopecurus pratensis without and with pollen of Dactyllis glomerata; (viii) pollen of Papaver orientale without and with pollen of Dactylis glomerata.

There were also fast changes (1 to 2 mm) in the fluorescence intensity of the pollen grains from weed species Artemisia vulgaris L. and Urtica dioica and day lily as well as of wind-pollinated, woody species Larix decidua and Betula verrucosa, which reflected the effects of volatile excretion (Table 8.5). When the microspores of weed species or woody species interacted, in both cases a 60 percent drop in light emission was observed. By contrast, day lily increased its fluorescence by 45 to 80 percent in the presence of weed pollen grains. According to Stanley and Linskens (1974), pollen has a noticeable smell. Terpenoids prevail among the volatile excretion (Egorov and Egofarova, 1971) and may influence the foreign pollen grains as well as attract insects.

Pollen-pollen antagonism, estimated by the ability to germinate, has been known since the 1930s for cultural plants of parterres (Bransheidt, 1930; Zanoni, 1930), whereas a mutual stimulation was first found for natural field plants by Golubinskii in 1946. Unlike the experiments needed for I to 24 h for the pollen tube growth, our experiments permitted testing the chemosensitivity of different pollen species in mixtures for I to 2 min, and can indicate compatible or incompatible species in phytocenosis. The ability to depress or stimulate fertilization by foreign pollen could be one of the determinants of the mutual existence of the species. Certainly, pollen of Papaver orientale and Hememcallis fulva, as parterre species, were either insensitive or weakly sensitive to pollen of field-grown, wind-pollinated Alopecurus pratensis L. and Dactylis glomerata. Only the fluorescence of Hemerocallis pollen was stimulated by pollen of weeds Artemisia vulgaris and Urtica dioica. In contrast, the pollen grains of wind-pollinated weeds and woody species were extremely sensitive to each other, decreasing their autofluorescence. The sensitivity between pollen of wind-pollinated meadow plants Alopecurus and Dactylis was marked, but without a strong correlation with their germination.

Fast processes observed as quick shifts in the fluorescence spectra or intensity should be associated with (I) metabolic processes, such as redox changes of NAD(P)H and flavines (Karnauchov 1978); (2) photodynamic changes of some photosensitive pigments of excreta and/or structures of cellular surface (Aucoin et al., 1992); and (3) free radical reactions, both in excreta and on the cellular surface.

Known photodynamic processes are connected with the allelochemicals excreted on the cellular surface such as furanocoumarins (Ceska et al., 1986), or located in surface glands, such as hypericin and juglone and its derivatives (Aucoin et al., 1992). Free radical reactions are most rapid. They are shown in plant excretion containing allelochemicals (Yurin et al., 1972) as spots of fluorescent pigment lipofuscin (Brooks and Csallany. 1978; Merzlyak, 1988), especially when in contact with ozone (Roshchina, 1996), on the pollen surface (Dodd and Ebert, 1971), or on the surface of seeds and leaves tested on final products. Free radicals and the products of free radical processes can contribute to visible light emission. Moreover, some substances such as hypericin, oleic acid, and berberine, may undergo both photodynamic and free radical reactions (Aucoin et al., 1992).

The fast changes in fluorescence at 430 to 470 nm from a plant surface may be due to NAD(P)H fluorescence. For instance, for 25 s the fluorescence at 460 nm in ascitic tumors decreased by 50 percent in the presence of fatty acids (Shwartsburd and Aslanidi, 1991).

TABLE 8.5 Fast Observations of Chemical Interactions of Dry Pollen Grains in Mixtures Estimated by the Fluorescence Intensity

Species of Pollen-Acceptor
of Volatile Excreta
Species of Pollen-Donor
of Volatile Excreta
Fluorescence Intensity of
Pollen-Acceptor at
475 nm (% of control)
Urtica dioica L. Artemisia vulgaris  L. 40 ± 1.0
Hemerocallis fulva L. Artemisia vulgaris L. 181 ± 2.3
Hemerocallis fulva L. Urtica dioica L. 145 ± 5.1
Larix decidua L. Betula verrucosa Ehrh. 45 ± 2.4

Golubinski (1946) discovered that a liberal amount of pollen on the stigma effectively stimulates their viability. The mixing of pollen of several varieties, induces notable interference, positive or negative.

Golubinski, I. N. 1946. The influence of pollen grain mixture and their density on their germination (Russian). Agrobiologia, 3; 39-70. Hort. Abstr. 91: (1948)

Zanoni, D.G., Antagonismo pollinico, Revista di Biologia, 12, 126, 1930.