S-RNase is the female determinant of self-incompatibility (SI) in pear (Pyrus bretschneideri). After translocation to the pollen tube, S-RNase degrades rRNA and induces pollen tube death in an S-haplotype-specific manner. In this study, we found that the actin cytoskeleton is a target of P. bretschneideri S-RNase (PbrS-RNase) and uncovered a mechanism that involves phosphatidic acid (PA) and protects the pollen tube from PbrS-RNase cytotoxicity. PbrS-RNase interacts directly with PbrActin1 in an S-haplotype-independent manner, causing the actin cytoskeleton to depolymerize and promoting programmed cell death in the self-incompatible pollen tube. Proline 156 of PbrS-RNase is essential for the PbrS-RNase-PbrActin1 interaction, and the actin cytoskeleton-depolymerizing function of PbrS-RNase does not require its RNase activity. PbrS-RNase cytotoxicity enhances the expression of phospholipase D (PbrPLDδ1), resulting in increased PA levels in the incompatible pollen tube. PbrPLDδ1-derived PA initially prevents depolymerization of the actin cytoskeleton elicited by PbrS-RNase and delays the SI signaling that leads to pollen tube death. This work provides insights into the orchestration of the S-RNase-based SI response, in which increased PA levels initially play a protective role in incompatible pollen, until sustained PbrS-RNase activity reaches the point of no return and pollen tube growth ceases.

Self-incompatibility (SI) is a widespread genetically determined system in flowering plants that prevents self-fertilization to promote gene flow and limit inbreeding. S-RNase-based SI is characterized by the arrest of pollen tube growth through the pistil. Arrested pollen tubes show disrupted polarized growth and swollen tips, but the underlying molecular mechanism is largely unknown. Here, we demonstrate that the swelling at the tips of incompatible pollen tubes in pear (Pyrus bretschneideri, Pbr) is mediated by the SI-induced acetylation of the soluble inorganic pyrophosphatase (PPA). PbrPPA5. Acetylation at Lys-42 of PbrPPA5 by the acetyltransferase GCN5-related N-acetyltransferase 1 (GNAT1) drives accumulation of PbrPPA5 in the nucleus, where it binds to the transcription factor PbrbZIP77, forming a transcriptional repression complex that inhibits the expression of the pectin methylesterase (PME) gene PbrPME44. The function of PbrPPA5 as a transcriptional repressor does not require its pyrophosphatase activity. Downregulating PbrPME44 resulted in increased levels of methyl esterified pectins in growing pollen tubes, leading to swelling at their tips. These observations suggest a mechanism for PbrPPA5-driven swelling at the tips of pollen tubes during the SI response. The targets of PbrPPA5 include genes encoding cell wall-modifying enzymes, which are essential for building a continuous sustainable mechanical structure for pollen tube growth.

Phosphoregulation of key sites on pollen soluble inorganic pyrophosphatases inhibits their catalytic responsiveness in concert with key intracellular events.

Polyamines (PAs) are essential metabolites in plants performing multiple functions during growth and development. Copper-containing amine oxidases (CuAOs) catalyse the catabolism of PAs and in Arabidopsis thaliana are encoded by a gene family. Two mutants of one gene family member, AtCuAOδ, showed delayed seed germination, leaf emergence, and flowering time. The height of the primary inflorescence shoot was reduced, and developmental leaf senescence was delayed. Siliques were significantly longer in mutant lines and contained more seeds. The phenotype of AtCuAOδ over-expressors was less affected. Before flowering, there was a significant increase in putrescine in AtCuAOδ mutant leaves compared to wild type (WT), while after flowering both spermidine and spermine concentrations were significantly higher than in WT leaves. The expression of GA (gibberellic acid) biosynthetic genes was repressed and the content of GA₁, GA₇, GA₈, GA₉, and GA₂₀ was reduced in the mutants. The inhibitor of copper-containing amine oxidases, aminoguanidine hydrochloride, mimicked the effect of AtCuAOδ mutation on WT seed germination. Delayed germination, reduced shoot height, and delayed flowering in the mutants were rescued by GA₃ treatment. These data strongly suggest AtCuAOδ is an important gene regulating PA homeostasis, and that a perturbation of PAs affects plant development through a reduction in GA biosynthesis.

A large number of different plant lines are produced and maintained in a typical plant research laboratory, both as seed stocks and in active growth. These collections need careful and consistent management to track and maintain them properly, and this is a particularly pressing issue in laboratories undertaking research involving genetic manipulation due to regulatory requirements. Researchers and PIs need to access these data and collections, and therefore an easy-to-use plant-oriented laboratory information management system that implements, maintains and displays the information in a simple and visual format would be of great help in both the daily work in the lab and in ensuring regulatory compliance.

Accumulation of reactive oxygen species (ROS) in the stigma is crucial for the sexual reproduction of flowering plants. However, the derivation of stigmatic production of ROS in pear is still largely unknown. Here, we identified a respiratory burst oxidase homolog (Rboh) protein in pear (Pyrus bretschneideri), PbRbohD1, which is required for ROS accumulation in the stigma. Knockdown of PbRbohD1 by the antisense oligodeoxynucleotide (as-ODN) technique resulted in significant reductions of stigma ROS levels. Interaction analysis indicated that the N-terminal region of PbRbohD1 interacted with the stigmatic Rho of plant (ROP) protein PbrROP2, and PbRbohD1 colocalized with PbrROP2 in the plasma membrane. PbrROP2 activated the PbRbohD1 protein, thereby stimulating the production of ROS. Knockdown of PbrROP2 in the stigma drastically reduced the ROS levels. Thus, during pear stigma development, PbrROP2 and PbRbohD1 work together to control the stigmatic ROS level. Reducing the stigmatic ROS level with a ROS scavenger, sodium salicylate (Na-SA), accelerated the growth of self-pollen tubes in the style and increased the fruit set rate. Our results uncover a signaling pathway that regulates the levels of stigmatic ROS, providing the physiological basis for improving pollination efficiency and fruit set rate.