Self S-RNase inhibits ABF-LRX signaling to arrest pollen tube growth to achieve self-incompatibility in pear.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

Inheritance of hetero-diploid pollen S-haplotype in self-compatible tetraploid Chinese cherry (Prunus pseudocerasus Lindl).

The breakdown of self-incompatibility, which could result from the accumulation of non-functional S-haplotypes or competitive interaction between two different functional S-haplotypes, has been studied extensively at the molecular level in tetraploid Rosaceae species. In this study, two tetraploid Chinese cherry (Prunus pseudocerasus) cultivars and one diploid sweet cherry (Prunus avium) cultivar were used to investigate the ploidy of pollen grains and inheritance of pollen-S alleles. Genetic analysis of the S-genotypes of two intercross-pollinated progenies showed that the pollen grains derived from Chinese cherry cultivars were hetero-diploid, and that the two S-haplotypes were made up of every combination of two of the four possible S-haplotypes. Moreover, the distributions of single S-haplotypes expressed in self- and intercross-pollinated progenies were in disequilibrium. The number of individuals of the two different S-haplotypes was unequal in two self-pollinated and two intercross-pollinated progenies. Notably, the number of individuals containing two different S-haplotypes (S1- and S5-, S5- and S8-, S1- and S4-haplotype) was larger than that of other individuals in the two self-pollinated progenies, indicating that some of these hetero-diploid pollen grains may have the capability to inactivate stylar S-RNase inside the pollen tube and grow better into the ovaries.

Reciprocal regulation of Ca²+-activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide.

• The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. • In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. • Heme (1 µM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 µM intracellular free Ca²+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. • These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.

Self-compatibility of 'Katy' apricot (Prunus armeniaca L.) is associated with pollen-part mutations.

Apricot (Prunus armeniaca L.) cultivars originated in China display a typical S-RNase-based gametophytic self-incompatibility (GSI). 'Katy', a natural self-compatible cultivar belonging to the European ecotype group, was used as a useful material for breeding new cultivars with high frequency of self-compatibility by hybridizing with Chinese native cultivars. In this work, the pollen-S genes (S-haplotype-specific F-box gene, or SFB gene) of 'Katy' were first identified as SFB1 and SFB (8), and the S-genotype was determined as S1 S8. Genetic analysis of 'Katy' progenies under controlled pollination revealed that the stylar S1-RNase and S8-RNase have a normal function in rejecting wild-type pollen with the same S-haplotype, while the pollen grains carrying either the SFB1 or the SFB8 gene are both able to overcome the incompatibility barrier. However, the observed segregation ratios of the S-genotype did not fit the expected ratios under the assumption that the pollen-part mutations are linked to the S-locus. Moreover, alterations in the SFB1 and SFB8 genes and pollen-S duplications were not detected. These results indicated that the breakdown of SI in 'Katy' occurred in pollen, and other factors not linked to the S-locus, which caused a loss of pollen S-activity. These findings support a hypothesis that modifying factors other than the S-locus are required for GSI in apricot.

Identification of hyperpolarization-activated calcium channels in apical pollen tubes of Pyrus pyrifolia.

The pollen tube has been widely used to study the mechanisms underlying polarized tip growth in plants. A steep tip-to-base gradient of free cytosolic calcium ([Ca(2+)](cyt)) is essential for pollen-tube growth. Local Ca(2+) influx mediated by Ca(2+)-permeable channels plays a key role in maintaining this [Ca(2+)](cyt) gradient. Here, we developed a protocol for successful isolation of spheroplasts from pollen tubes of Pyrus pyrifolia and identified a hyperpolarization-activated cation channel using the patch-clamp technique. We showed that the cation channel conductance displayed a strong selectivity for divalent cations, with a relative permeability sequence of barium (Ba(2+)) approximately Ca(2+) > magnesium (Mg(2+)) > strontium (Sr(2+)) > manganese (Mn(2+)). This channel conductance was selective for Ca(2+) over chlorine (Cl(-)) (relative permeability P(Ca)/P(Cl) = 14 in 10 mm extracellular Ca(2+)). We also showed that the channel was inhibited by the Ca(2+) channel blockers lanthanum (La(3+)) and gadolinium (Gd(3+)). Furthermore, channel activity depended on extracellular pH and pollen viability. We propose that the Ca(2+)-permeable channel is likely to play a role in mediating Ca(2+) influx into the growing pollen tubes to maintain the [Ca(2+)](cyt) gradient.

[The effect of G protein regulator on pollen germination and [Ca2+]i variation in Pyrus serotina Rehd. pollen].

The effects of the cholera toxin (CTX) and pertussis toxin (PTX), which can activate and antagonize respectively the heterotrimeric G protein, on pollen germination, pollen tube growth and the dynamic of cytosolic free calcium concentration ([Ca(2+)]i) in pear pollen were investigated using Laser Confocal Scanning Microscope (LCSM). The results show that CTX could stimulate pollen germination and tube growth while PTX had the opposite effect. CTX and PTX had marked influence on the dynamic of pollen [Ca(2+)]i. The CTX treatment had no significant effect on the changes in pollen [Ca(2+)]i during pollen germination, but induced specific signaling of "calcium transient" in pollen. Treatment with PTX caused decline of pollen [Ca(2+)]i in the first 18 min after treatment. These data suggested that the regulation of pollen germination and tube growth in Pyrus serotina may involve in heterotrimeric G protein, which can stimulate specific change of pollen [Ca(2+)]i.