Characterization of a legumain/vacuolar processing enzyme and YVADase activity in Papaver pollen.

Legumains, also known as Vacuolar Processing Enzymes (VPEs) have received considerable attention recently, as they share structural properties with mammalian caspase-1 and exhibit YVADase/caspase-1-like cleavage activity. Although many legumains have been cloned, knowledge about their detailed characteristics and intracellular localization is relatively limited. We previously identified several caspase-like activities activated by self-incompatibility (SI) in pollen; a DEVDase was required for programmed cell death (PCD), but YVADase was not (Bosch and Franklin-Tong in Proc Natl Acad Sci USA 104:18327-18332, 2007; Thomas and Franklin-Tong in Nature 429:305-309, 2004). Here we report identification of a legumain/VPE from Papaver rhoeas pollen (PrVPE1) that binds to the DEVD tetrapeptide, a signature substrate for caspase-3. A detailed characterization of the recombinant PrVPE1 cleavage activity revealed that, like other VPEs, it has YVADase activity and requires an acidic pH for activity. Unlike other legumain/VPEs, it also exhibits DEVDase and IETDase activities and apparently does not require processing for activity. The pollen-expressed PrVPE1 localizes to a reticulate compartment resembling the vacuole. Examination of YVADase activity using live-cell imaging of pollen tubes revealed YVADase activity in mitochondria of growing pollen tubes. The unexpected features of PrVPE1, together with evidence for YVADase activity in plant mitochondria, indicate that VPEs, YVADases, their localization and functions in plant cells merit further investigation.

Identification of the pollen self-incompatibility determinant in Papaver rhoeas.

Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility is an important mechanism used in many species to prevent inbreeding; it is controlled by a multi-allelic S locus. 'Self' (incompatible) pollen is discriminated from 'non-self' (compatible) pollen by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca(2+)-dependent signalling network, resulting in pollen inhibition and programmed cell death. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS (Papaver rhoeas pollen S), from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single-copy gene linked to the pistil S gene (currently called S, but referred to hereafter as PrsS for Papaver rhoeas stigma S determinant). Sequence analysis indicates that PrsS and PrpS are equally ancient and probably co-evolved. PrpS encodes a novel approximately 20-kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver self-incompatibility system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between self and non-self, which also include histocompatibility systems in primitive chordates and vertebrates.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen.

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.