Supplementary Information.
Supplementary Fig S1. Model for sPPase phosphorylation, effect on activity and biological consequences.
(a)Proposed role for Pr-p26.1a/b sPPases in normal pollen tube growth (left).
sPPases important enzymes that catalyse hydrolysis of inorganic pyrophosphate (PPi), providing the driving force for many metabolic reactions. During biopolymer synthesis, PPi is generated and hydrolyzed to inorganic phosphate (2 Pi); this provides a thermodynamic pull favouring further biosynthesis. Normal pollen tube growth, which involves extensive biosynthesis of new membrane and cell wall, requires soluble inorganic pyrophosphatase (sPPase) activity. We show, using antisense oligonucleotides to conserved Pr-p26.1a/b sequences, that Pr-p26.1a/b plays a functional role that is critical for pollen tube growth, as the antisense oligonucleotides cause inhibition of pollen tube growth.
(b)Proposed role for Pr-p26.1a/b sPPases during SI in incompatible pollen tube inhibition (right).
SI stimulates increases in cytosolic free calcium [Ca2+]i which activates a calcium-dependent protein kinase (CDPK), which phosphorylates Pr-p26.1a/b. Both calcium and phosphorylation inhibit sPPase activity. As sPPases are important enzymes involved in regulating biosynthesis, this provides a mechanism to inhibit pollen tube growth. In this way, SI-induced inhibition of sPPase activity, which is predicted to prevent hydrolysis of PPi, results in accumulation of PPi andso causes a reduction in the biosynthetic capability of the incompatible pollen tube, thereby contributing to inhibition of growth.
Supplementary Fig S2. Deduced amino acid sequences of the Pr-p26.1a/b cDNAs.
The original 15 amino acid peptide sequence obtained from p26.1 is indicated in bold italics. Amino acid identities (black shading), similarities (grey shading). 17 highly conserved amino acids common to sPPases (black triangles); sPPase signature DXDXXD (black circles). Peptide sequences obtained from phosphoaffinity purified p26.1 (bold). Predicted putative phosphorylation sites (*).
Supplementary Fig S3.
sPPase activities in different tissues from Papaver rhoeas
Cytosolic extracts from germinated pollen (GP) and hydrated pollen (HP) showed much higher levels of sPPase activity than cytosolic extracts from other tissues (mature leaf (ML), immature leaf (IL), stem (S), root (R), stigma (St), ovary (O), immature anthers (IA) fromPapaver rhoeas. Data are means ± s.e.m, n=6 except GP and ML, n=3).
Supplementary Fig S4.
Antisense oligonucleotides show that Pr-p26.1a/b play a key role in pollen tube growth
Antisense oligonucleotides in the presence of cytofectin (as-ODN; top panel) resulted in strong inhibition of pollen tube growth, while sense oligonucleotides in the presence of cytofectin (s-ODN; bottom panel) did not. This demonstrates that Pr-p26.1 is crucial for pollen tube growth.
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