Live-cell imaging of early events following pollen perception in self-incompatible Arabidopsis thaliana

Abstract

Early events occurring at the surface of the female organ are critical for plant reproduction, especially in species with a dry stigma. After landing on the stigmatic papilla cells, the pollen hydrates and germinates a tube, which penetrates the cell wall and grows towards the ovules to convey the male gametes to the embryo sac. In self-incompatible species within the Brassicaceae, these processes are blocked when the stigma encounters an incompatible pollen. Based on the generation of self-incompatible Arabidopsis lines and by setting up a live imaging system, we showed that control of pollen hydration has a central role in pollen selectivity. The faster the pollen pumps water from the papilla during an initial period of 10 min, the faster it germinates. Furthermore, we found that the self-incompatibility barriers act to block the proper hydration of incompatible pollen and, when hydration is promoted by high humidity, an additional control prevents pollen tube penetration into the stigmatic wall. In papilla cells, actin bundles focalize at the contact site with the compatible pollen but not with the incompatible pollen, raising the possibility that stigmatic cells react to the mechanical pressure applied by the invading growing tube.

The molecular signatures of compatible and incompatible pollination in Arabidopsis

“…This suggests that key molecules required for the incompatibility reaction are likely to be already present in the stigma. This may be consistent with the observation that the pollen rejection response in A. thaliana lines exhibiting a restored incompatibility system is extremely rapid and occurs within minutes [ 3 , 12 ].…”

“…Early work showed that self-incompatibility can be restored in the self-fertile species A. thaliana by reintroducing a functional SRK-SCR gene pair isolated from its close self-incompatible relative A. lyrata [ 21 , 22 ]. To analyze both compatible and incompatible reactions and take advantage of nucleotide polymorphisms between A. thaliana accessions, previously, we generated two transgenic lines: one in the Col-0 background expressing the SRK gene from the A. lyrata S 14 haplotype (Col-0/ SRK14 ) and the other in C24 background expressing the SCR gene from the same S -haplotype (C24/ SCR14 ) [ 3 ]. Both lines were self-fertile but when stage 13-14E (according to [ 23 ]) Col-0/ SRK14 stigmas were pollinated with C24/ SCR14 pollen, a strong incompatible reaction was observed as deduced from the absence of pollen tube in the stigma, 1 hour after pollen deposition (Fig.…”

“…Using these two Arabidopsis lines, we performed a compatible and incompatible pollination kinetics focusing on two time-points of the interaction to identify genes whose expression is modified following pollen-stigma interaction. We selected an early stage (10 min after pollen deposition), which corresponds to the start of pollen grain hydration [ 3 ], and a later stage (one hour after pollination) when pollen tubes reach the base of the stigma. We sequenced mRNAs extracted from pollinated stigmas at 10, and 60 min after compatible (Col-0/ SRK14 x C24) pollination (C10, C60, respectively) and incompatible (Col-0/ SRK14 x C24/ SCR14 ) pollination (I10, I60, respectively) (Fig.…”

“…Nevertheless, it is worth noting that the two upmost enriched GO categories are related to water or fluid movements (water transport (GO:0006833), fluid transport (GO:0042044)). As Pollen hydration is highly controlled at the surface of Brassicaceae stigma [ 3 ], we may assume that these GO term categories reflect the regulation of water fluxes from the stigmatic cells towards the incompatible pollen.…”

“…Following compatible pollination, the dry pollen grain hydrates on the stigma papilla and ultimately germinates, producing a tube that penetrates the wall of the stigmatic cell and grows down to convey the male gametes towards the ovules for fertilization [ 1 , 2 ]. By contrast, when a pollen grain is recognized as incompatible, it fails to hydrate properly and shows defective germination [ 3 ]. This rejection mechanism is initiated by a ligand-receptor interaction and is genetically controlled by a single polymorphic locus, called the S -locus [ 4 ].…”