School of Environmental and Rural Science, University of New England, Armidale, Australia.
CSIRO, Clunies Ross St., Acton, ACT, Australia.
Sci Rep. 2021 Mar 23;11(1):6635. doi: 10.1038/s41598-021-85887-y.
Insects are essential for the reproduction of pollinator-dependent crops and contribute to the pollination of 87% of wild plants and 75% of the world's food crops. Understanding pollen flow dynamics between plants and pollinators is thus essential to manage and conserve wild plants and ensure yields are maximized in food crops. However, the determination of pollen transfer in the field is complex and laborious. We developed a field experiment in a pollinator-dependent crop and used high throughput RNA sequencing (RNA-seq) to quantify pollen flow by measuring changes in gene expression between pollination treatments across different apple (Malus domestica Borkh.) cultivars. We tested three potential molecular indicators of successful pollination and validated these results with field data by observing single and multiple visits by honey bees (Apis mellifera) to apple flowers and measured fruit set in a commercial apple orchard. The first indicator of successful outcrossing was revealed via differential gene expression in the cross-pollination treatments after 6 h. The second indicator of successful outcrossing was revealed by the expression of specific genes related to pollen tube formation and defense response at three different time intervals in the stigma and the style following cross-pollination (i.e. after 6, 24, and 48 h). Finally, genotyping variants specific to donor pollen could be detected in cross-pollination treatments, providing a third indicator of successful outcrossing. Field data indicated that one or five flower visits by honey bees were insufficient and at least 10 honey bee flower visits were required to achieve a 25% probability of fruit set under orchard conditions. By combining the genotyping data, the differential expression analysis, and the traditional fruit set field experiments, it was possible to evaluate the pollination effectiveness of honey bee visits under orchards conditions. This is the first time that pollen-stigma-style mRNA expression analysis has been conducted after a pollinator visit (honey bee) to a plant (in vivo apple flowers). This study provides evidence that mRNA sequencing can be used to address complex questions related to stigma-pollen interactions over time in pollination ecology.
昆虫对于依赖传粉者的作物的繁殖至关重要，并且有助于授粉 87%的野生植物和 75%的世界粮食作物。因此，了解植物与传粉者之间的花粉流动动态对于管理和保护野生植物以及确保粮食作物产量最大化至关重要。然而，在田间确定花粉转移是复杂且费力的。我们在一种依赖传粉者的作物中进行了田间实验，并使用高通量 RNA 测序（RNA-seq）通过测量授粉处理之间的基因表达变化来量化花粉流动，该实验跨越了不同的苹果（Malus domestica Borkh.）品种。我们测试了三种成功授粉的潜在分子指标，并通过观察蜜蜂（Apis mellifera）对苹果花的单次和多次访问以及在商业苹果园中测量坐果率，用田间数据验证了这些结果。成功异交的第一个指标是通过 6 小时后异交授粉处理中的差异基因表达揭示的。成功异交的第二个指标是通过在异交授粉后柱头和花柱中与花粉管形成和防御反应相关的特定基因的表达在三个不同时间间隔揭示的（即 6、24 和 48 小时后）。最后，可以在异交授粉处理中检测到与供体花粉特异性的基因分型变体，这提供了成功异交的第三个指标。田间数据表明，蜜蜂对一朵花的 1 次或 5 次访问是不够的，并且至少需要 10 次蜜蜂对花的访问才能在果园条件下达到 25%的坐果概率。通过结合基因分型数据、差异表达分析和传统的坐果田间实验，可以评估在果园条件下蜜蜂访问的授粉效果。这是第一次在植物（活体苹果花）上进行传粉者访问（蜜蜂）后进行花粉-柱头-花柱 mRNA 表达分析。本研究为使用 mRNA 测序解决授粉生态学中随时间推移柱头-花粉相互作用的复杂问题提供了证据。
