Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir

Abstract

Pollen of larch (Larix × marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix × marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.

Application of proteomics to the study of pollination drops

“…Another line of research arising from such protein surveys is comparative biology. Proteomes of pollination drops show similarities (Nepi et al, 2009; Coulter et al, 2012; von Aderkas et al, 2012) with other extracellular exudates, such as root secretions (Hawes et al, 2011) and nectar (Thornburg et al, 2003; Heil, 2011). …”

“…This new knowledge has changed how we view the first contact of pollen and ovule in gymnosperms. Previously, pollination drops have been treated as mere receiving agents of pollen (Singh, 1978), but we now know, thanks to proteomics, that different species have unique pollination drop protein composition, which influences germination of conspecific vs. heterospecific pollen (von Aderkas et al, 2012). Pollen germinates in a relatively microbe-free environment because drops in some species have a number of antimicrobial protein classes present (Poulis et al, 2005; O’Leary et al, 2007; Wagner et al, 2007).…”

“…To prove function, we have recently shown that an antifungal chitinase identified from Pseudotsuga drops exhibits enzyme activity both in situ and in vitro (Coulter et al, 2012). In addition, we have shown that drop-identified invertases are able to alter the carbohydrate composition of drops in situ, creating different osmotic environments in which pollen germinates (von Aderkas et al, 2012). Protein identifications were also used as a starting point to devise experiments to prove that these proteins originate from nucellus (Poulis et al, 2005).…”

“…In particular, the active biological role of proteins in pollination drops has only recently come to light (von Aderkas et al, 2012). Drops are able to modify extracellular carbohydrate composition with secreted invertases, which favors conspecific pollen germination over heterospecific pollen germination (von Aderkas et al, 2012). Drops also have functional chitinases that play a role in ovule defense (Coulter et al, 2012).…”

“…Quantification of individual proteins has not yet been attempted using targeted proteomics methods such as multiple reaction monitoring (Picotti and Aebersold, 2012; Maiolica et al, 2012). Proteomics investigations have been followed up by biochemical assays to verify in situ and in vitro functionality of pollination drop enzymes (Coulter et al, 2012; von Aderkas et al, 2012). Discovery of proteins is accomplished by generating peptides whose sequences can be used to query protein databases providing unequivocal identification when sequence information is sufficient (Steen and Mann, 2004).…”