Self-pollen Growth Research Articles

Mechanisms of S-RNase-based self-incompatibility.

Abstract Gametophytic self-incompatibility (GSI), a genetic mechanism that acts to prevent self-fertilization in many angiosperms, is based on the ability of the pistil to selectively inhibit growth of self pollen while allowing the growth of non-self pollen. In the most widespread variant of GSI (studied most thoroughly in the Solanaceae, Rosaceae and Plantaginaceae), a style-expressed ribonuclease (the S-RNase) is imported into both self and non-self pollen tubes. In pollen tubes recognized as self, the S-RNase degrades RNA, slowing protein synthesis and growth of the pollen tube, whereas in non-self pollen tubes the action of the S-RNase is inhibited and growth continues normally. Recognition of self versus non-self in pollen appears to be mediated through the action of the recently identified SLF/SFB protein. Protein interaction studies suggest that SLF/SFB acts via a modified SCFSLF E3 ubiquitin ligase complex. Several questions remain, however, about the precise mechanism by which the S-RNase remains inactive in non-self pollen tubes. Experiments tracking the fate of protein components of self-incompatibility in pollinated styles suggest that in compatible pollinations, the S-RNase remains sequestered in a membrane-bounded vacuolar compartment, while it is released in incompatible pollen tubes. Other experiments suggest that ubiquitination, and possibly degradation, of S-RNases occurs preferentially in incompatible pollen compared with compatible pollen. Finally, the analysis of SFB mutants in the Rosaceae, along with investigations into the breakdown of GSI in tetraploid plants, suggest that details of the GSI response may differ in the Rosaceae compared with the Solanaceae and Plantaginaceae.

Suitability of four different methods to identify self-compatible seedlings in an almond breeding programme

SummaryObtaining self-compatible almond cultivars from controlled crosses has become one of the most important aims in almond breeding programmes. Self-compatible seedlings from such crosses have been identified by different methods. Determination of fruit set in bagged branches and observing self-pollen growth along the pistil by fluorescence microscopy are methods traditionally used. Non-equilibrium pH gradient electro-focusing (NEPHGE) of stylar S-RNases is a more recent method, which reveals self-compatibility by partial or total absence of ribonuclease activity in heterozygous and homozygous self-compatible individuals respectively. To date, the most recent method is S-allele PCR, which identifies the presence of the Sf allele in genomic DNA. In this work, reliability, ease of performance, economic cost and time spent in obtaining the results of these four methods were compared in a progeny of 74 seedlings. The results showed that, considering its moderate cost and time spent, NEPHGE of stylar S-RNases was the most convenient method. However, as PCR of S-alleles can be applied in the first year to plantlets in the nursery, an early selection of self-compatible seedlings is possible. On the other hand, bagging is essential in a further stage of the selection process, thus ensuring good yields in single-cultivar almond orchards.

The male determinant of self‐incompatibility in Brassica oleracea is located in the pollen coating

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea. In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an ‘extension’ of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of ‘self’ coating (i.e. that with the same S‐haplotype as the stigma) prevent the success of a compatible cross pollination, but a ‘cross’ coating (i.e. that with a different S‐haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen‐held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an Mr10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine‐rich proteins (PCP‐A: Pollen Coat Proteins‐class A)—one of which is known to bind to stigmatically‐expressed components of the S‐locus in Brassica. Introduction of the PCP‐A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP‐A protein family.

ROLE OF CALCIUM IN THE CALLOSE RESPONSE OF SELF‐POLLINATED BRASSICA STIGMAS

In Brassica oleracea, sporophytic self‐incompatibility prevents germination of self pollen, or normal growth of self pollen tubes. After self‐pollination, the papillae of stigmas synthesize callose. The role of Ca++ in the formation of stigmatic callose was tested by adding compounds that interact with Ca++ to suspensions of pollen that were known to induce callose formation in self stigmas. The calcium channel antagonist, lanthanum, and the calcium chelating agent, EGTA, reduced or abolished the callose response to self‐pollen suspensions. In the presence of Ca++, the calcium ionophore, A23187, induced callose in stigmatic papillae when added to pollen suspensions, or alone. Therefore, callose deposition in response to incompatible pollinations appears to be a calcium‐dependent process. Pretreatment of pistils with 100 μm 2‐deoxy‐D‐glucose abolished the callose response to self‐pollination, while self pollen remained inhibited and cross pollen grew normally in treated pistils. Thus, callose formation in the stigma is not an essential part of the self‐incompatibility mechanism preventing the growth of self pollen in Brassica.

Role of Calcium in the Callose Response of Self-Pollinated Brassica Stigmas

In Brassica oleracea, sporophytic self-incompatibility prevents germination of self pollen, or normal growth of self pollen tubes. After self-pollination, the papillae of stigmas synthesize callose. The role of Ca++ in the formation of stigmatic callose was tested by adding compounds that interact with Ca++ to suspensions of pollen that were known to induce callose formation in self stigmas. The calcium channel antagonist, lanthanum, and the calcium chelating agent, EGTA, reduced or abolished the callose response to self-pollen suspensions. In the presence of Ca++, the calcium ionophore, A23187, induced callose in stigmatic papillae when added to pollen suspensions, or alone. Therefore, callose deposition in response to incompatible pollinations appears to be a calcium-dependent process. Pretreatment of pistils with 100 μm 2-deoxy-D-glucose abolished the callose response to self-pollination, while self pollen remained inhibited and cross pollen grew normally in treated pistils. Thus, callose formation in the stigma is not an essential part of the self-incompatibility mechanism preventing the growth of self pollen in Brassica.

Differential Pollen Tube Success in Geranium caespitosum

Geranium caespitosum is self-compatible, yet selfing decreases both seed number and seed weight compared to outcrossing. A mechanism that inhibits the growth of self pollen would reduce losses in fitness from selfing and could be detected by comparing the number of pollen tubes entering ovules following self- and cross-pollination. Pistils receiving either self- or cross-pollen were each held for 2 h or 24 h and then prepared for fluorescence microscopy. After 2 h, fewer self- than cross-pollen tubes entered ovules. Differences between the number of self- and cross-pollen tubes entering ovules diminished after 24 h, indicating that many self-pollen tubes were delayed, but not excluded, from entering micropyles. Fluorescence microscopy confirmed that many pollen tubes are aborted in a specific region of transmitting tissue within the ovary. Thus, a labile mechanism favors cross-pollen but allows selfing when pollinators or neighboring plants are scarce.