Waterlily study links pollen rejection to reactive oxygen and two interacting proteins

A barrier before fertilization

Waterlily breeders have long sought to combine desirable traits from hardy and tropical varieties. The source paper describes one ambition: bringing tropical waterlilies’ blue flowers together with cold-resistant traits. Yet crosses between these groups can produce few seeds or none at all.

In a bioRxiv preprint posted on November 2, 2026, Jing Cao and colleagues investigate a barrier at the stigma, the flower structure that receives pollen. Their results connect failed pollen germination with increased reactive oxygen species, or ROS, and implicate two proteins: NcSnRK1 and NcRbohC1. The work has not been certified by peer review.

The researchers used the hardy waterlily ‘Arc-EN-Ciel’ as the female parent and the tropical ‘Nine Grade Blue’ as the pollen donor. They compared this cross with self-pollination of ‘Arc-EN-Ciel’.

Six hours after self-pollination, they observed abundant pollen tubes growing into the stigma. In the cross-pollinated flowers, pollen grains adhered to the stigma but did not germinate. That contrast places the observed obstacle before fertilization, rather than at a later stage of seed development.

Changing the chemical environment

ROS are chemically reactive oxygen-containing molecules. The study focuses on whether their balance in the stigma influences pollen germination.

Using fluorescent staining, the researchers detected significantly higher ROS levels in cross-pollinated stigmas than in self-pollinated stigmas half an hour after pollination. They then tested whether changing that chemical environment altered pollen behavior.

Adding the antioxidant dimethylthiourea, or DMTU, to the stigma fluid before cross-pollination reduced the ROS signal and allowed many pollen grains to germinate. Conversely, adding a xanthine/xanthine oxidase treatment before self-pollination increased ROS and inhibited germination.

These opposing results support the authors’ conclusion that ROS participates in regulating pollen germination in this experimental system. The evidence goes beyond an association between high ROS and failed germination: treatments that changed ROS levels also changed the pollen response.

A further treatment used DPI, an inhibitor of NADPH oxidase, an enzyme involved in ROS production. It lowered stigma ROS levels and allowed cross-pollen to germinate. The authors interpret this as evidence that NADPH oxidase is a major ROS source in the waterlily stigma.

Two candidate proteins

Earlier analyses of gene activity and proteins had highlighted NcRbohC1 as a candidate involved in stigma ROS regulation. Localization experiments in tobacco leaves placed this protein at the cell membrane.

To test its function, the researchers temporarily reduced or increased NcRbohC1 gene expression in waterlily plants using a virus-based system. Following cross-pollination, reducing its expression lowered ROS and permitted pollen germination. Increasing its expression was associated with high ROS and a failure to germinate. In self-pollination experiments, the overexpression group also had fewer germinating pollen grains than the interference group.

The team next searched for proteins that interact with the N-terminal region, one end of NcRbohC1. They identified NcSnRK1. Three approaches—yeast two-hybrid testing, luciferase complementation in tobacco leaves, and a biochemical pull-down assay—supported an interaction between NcSnRK1 and that region of NcRbohC1.

Temporarily changing NcSnRK1 expression produced a similar pattern. Reducing its expression improved cross-pollen germination and lowered ROS relative to overexpression. In self-pollinated flowers, overexpression likewise reduced germination compared with interference.

A proposed mechanism, not a finished solution

The authors propose that NcSnRK1 activates NcRbohC1 by phosphorylation, the addition of phosphate groups at sites near its N-terminal end. In their model, this triggers a ROS burst in stigma surface cells, disrupts the local ROS balance and inhibits pollen germination.

That phosphorylation step is an inference. The reported interaction assays support protein binding, but the study does not report direct identification or experimental verification of the proposed phosphorylation sites. Its discussion draws partly on previous work describing a related mechanism in tomato.

The breeding implications also need to be kept within the experiments’ scope. Restoring pollen germination is not the same as demonstrating successful fertilization, seed production or a new hybrid variety; those outcomes are not reported for the rescue treatments. The main cross-pollination comparison also uses one named parental combination.

The preprint therefore offers evidence for a ROS-linked early reproductive barrier and a candidate protein partnership, rather than a demonstrated breeding remedy. It points toward a mechanism that could inform efforts to overcome waterlily crossing barriers while leaving key molecular and reproductive outcomes unresolved.