Pistil Biology of ‘WA 38’ Apple and Effect of Pollen Source on Pollen Tube Growth and Fruit Set

‘WA 38’ (‘Enterprise’ × ‘Honeycrisp’) is an apple variety that is characterized by a peculiar self-thinning trait in which most of the fruitlets naturally shed within the first 8 weeks after bloom, leaving some clusters empty, but most with 1–2 apples. This study aimed to investigate potential causes for the relatively low fruit set observed in ‘WA 38’ by investigating its flower biology. This study comprised three objectives: (1) To characterize the effective pollination period (EPP) of ‘WA 38’ by studying stigmatic receptivity, pollen tube growth, and ovule longevity in ‘WA 38’ flowers, (2) to compare the pollen tube growth of 5 fully compatible pollinizer varieties in ‘WA 38’ pistils, and (3) to evaluate fruit and seed set resulting from controlled pollinations with 5 fully compatible pollinizer varieties. The results showed ‘WA 38’ EPP was approximately 3.2 days in 2019 and 1.4 days in 2020, and that differences in pollen sources did not attribute significant differences in fertility in ‘WA 38’ flowers. The results of this study suggest mechanisms other than pollination and fertilization, such as competition among fruitlets within a cluster or hormone signaling, may have a stronger impact on ‘WA 38’ fruitlet abscission.

Transcriptome Analysis of Triple Mutant for OsMADS62, OsMADS63, and OsMADS68 Reveals the Downstream Regulatory Mechanism for Pollen Germination in Rice (Oryza sativa)

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

Reciprocal intercompatibility between Byrsonima Rich. exKunth species (Malpighiaceae), in an urban ecotonal fragment of Semi-Deciduous Seasonal Forest and Cerrado

Byrsonima species present floral monomorphism and sharing visitor/pollinator guild. These traits suggest the hypothesis evaluation of reciprocal inter-incompatibility between two syntopic Byrsonima species: B. pachyphylla and B. crassifolia. Pollen tube growth with similar behavior was observed in the stigmatic surface, pistil canal, ovary and micropylar channel to both species. In addition, partial self-incompatibility in self-pollination with greater fruiting in autogamy was observed. Cross-pollination and self-pollination coexist, and reciprocal intercompatibility occurs. Prezygotic isolation mechanisms are unlikely by the absence of abnormal pollen tubes, higher fruiting production and absence of hybrids in the study site. Las especies de Byrsonima presentan monomorfismo floral y comparten gremio de visitantes/polinizadores. Estos rasgos sugieren la evaluación de hipótesis de interincompatibilidad recíproca entre dos especies sintópicas de Byrsonima: B. pachyphylla y B. crassifolia. Se observó el crecimiento del tubo polínico con comportamiento similar en la superficie estigmática, canal del pistilo, ovario y canal micropilar de ambas especies. Además, se describió la autoincompatibilidad parcial en la autopolinización con mayor fructificación en la autogamia. La polinización cruzada y la autopolinización coexisten y se produce una intercompatibilidad recíproca. Los mecanismos de aislamiento precigóticos son improbables por la ausencia de tubos polínicos anormales, producción de frutos y ausencia de híbridos en la área de estudio.

Reproductive barriers in cassava: Factors and implications for genetic improvement

Cassava breeding is hampered by high flower abortion rates that prevent efficient recombination among promising clones. To better understand the factors causing flower abortion and propose strategies to overcome them, we 1) analyzed the reproductive barriers to intraspecific crossing, 2) evaluated pollen-pistil interactions to maximize hand pollination efficiency, and 3) identified the population structure of elite parental clones. From 2016 to 2018, the abortion and fertilization rates of 5,748 hand crossings involving 91 parents and 157 progenies were estimated. We used 16,300 single nucleotide polymorphism markers to study the parents’ population structure via discriminant analysis of principal components, and three clusters were identified. To test for male and female effects, we used a mixed model in which the environment (month and year) was fixed, while female and male (nested to female) were random effects. Regardless of the population structure, significant parental effects were identified for abortion and fertilization rates, suggesting the existence of reproductive barriers among certain cassava clones. Matching ability between cassava parents was significant for pollen grains that adhered to the stigma surface, germinated pollen grains, and the number of fertilized ovules. Non-additive genetic effects were important to the inheritance of these traits. Pollen viability and pollen-pistil interactions in cross- and self-pollination were also investigated to characterize pollen-stigma compatibility. Various events related to pollen tube growth dynamics indicated fertilization abnormalities. These abnormalities included the reticulated deposition of callose in the pollen tube, pollen tube growth cessation in a specific region of the stylet, and low pollen grain germination rate. Generally, pollen viability and stigma receptivity varied depending on the clone and flowering stage and were lost during flowering. This study provides novel insights into cassava reproduction that can assist in practical crossing and maximize the recombination of contrasting clones.

Polyamines Involved in Regulating Self-Incompatibility in Apple

Apple exhibits typical gametophytic self-incompatibility, in which self-S-RNase can arrest pollen tube growth, leading to failure of fertilization. To date, there have been few studies on how to resist the toxicity of self-S-RNase. In this study, pollen tube polyamines were found to respond to self-S-RNase and help pollen tubes defend against self-S-RNase. In particular, the contents of putrescine, spermidine, and spermine in the pollen tube treated with self-S-RNase were substantially lower than those treated with non-self-S-RNase. Further analysis of gene expression of key enzymes in the synthesis and degradation pathways of polyamines found that the expression of DIAMINE OXIDASE 4 (MdDAO4) as well as several polyamine oxidases such as POLYAMINE OXIDASES 3 (MdPAO3), POLYAMINE OXIDASES 4 (MdPAO4), and POLYAMINE OXIDASES 6 (MdPAO6) were significantly up-regulated under self-S-RNase treatment, resulting in the reduction of polyamines. Silencing MdPAO6 in pollen tubes alleviates the inhibitory effect of self-S-RNase on pollen tube growth. In addition, exogenous polyamines also enhance pollen tube resistance to self-S-RNase. Transcriptome sequencing data found that polyamines may communicate with S-RNase through the calcium signal pathway, thereby regulating the growth of the pollen tubes. To summarize, our results suggested that polyamines responded to the self-incompatibility reaction and could enhance pollen tube tolerance to S-RNase, thus providing a potential way to break self-incompatibility in apple.