STIL, a peculiar molecule from styles, specifically dephosphorylates the pollen receptor kinase LePRK2 and stimulates pollen tube growth in vitro

Abstract

Background: LePRK1 and LePRK2 are two pollen receptor kinases localized to the plasma membrane, where they are present in a high molecular weight complex (LePRK complex). LePRK2 is phosphorylated in mature and germinated pollen, but is dephosphorylated when pollen membranes are incubated with tomato or tobacco style extracts.

Results: Here we show that LePRK2 dephosphorylation is mediated by a heat-, acid-, base-, DTT- and protease-resistant component from tobacco styles. Using LePRK2 phosphorylation as a tracking assay for purification, style exudates were subjected to chloroform extraction, anionic exchange, and C18 reverse-phase chromatography columns. We finally obtained a single ~3,550 Da compound (as determined by UV-MALDI-TOF MS) that we named STIL (for Style Interactor for LePRKs). STIL increased pollen tube lengths of in vitro germinated pollen in a dose-dependent manner.

Conclusion: We propose that the LePRK complex perceives STIL, resulting in LePRK2 dephosphorylation and an increase in pollen tube growth.

Figure 1

Characterization of the LePRK2 dephosphorylating activity in tobacco style extracts. A, Pollen microsomal fractions (15 μg) were incubated for 10 min with [gamma-³²P]-ATP in buffer without (lane 1) or with (lane 2) tobacco style extract proteins (340 μg), or with (lane 3) tomato style exudate proteins (190 μg), then separated by SDS-PAGE, blotted onto nitrocellulose and subjected to autoradiography (top panel, ³²P), then incubated with anti-LePRK2 antibody (bottom panel, Western Blot). The position of LePRK2 is indicated by arrows. B, Dephosphorylation activity after chloroform extraction of style extracts. Lane 1, aqueous phase; lane 2, interface; and lane 3, organic phase. The position of LePRK2 is indicated by an arrow.

Figure 2

Purification of STIL. A, Chromatograph of the C18 reverse-phase semi-preparative column. The sample was loaded with 6% acetonitrile (MeCN) and eluted with 50% and 100% acetonitrile (indicated by dashed lines). Fractions were assayed for LePRK2 dephosphorylation activity (inset). The position of LePRK2 is indicated by an arrow. B, Purification protocol used for the isolation of STIL from tobacco stigma/style exudates. C, Chromatograph of the second Mono Q column. Abs280 nm (left vertical axis, solid line) and % of Buffer (100% Buffer corresponding to 1 M NH₄HCO₃; right vertical axis, dashed line). Fractions 3 to 27 were assayed for LePRK2 dephosphorylation activity (inset).

Figure 3

UV-MALDI-TOF MS spectrum of pure STIL. STIL is a 3,548.39 Da entity (spectrum corresponds to fractions 14 and 15, Fig. 2C).

Figure 4

STIL is labile to microwave-assisted acid hydrolysis. A, Dilution series of the LePRK2 dephosphorylating activity of pure STIL. Lane 1, untreated pollen microsomal fraction. Lane 2 was treated with 4.8 × 10^-2 Abs280 units of tobacco style extract. Serial dilutions of purified STIL (lanes 3 through 14, from Fig. 2C) were used in the dephosphorylation assay; B, Pollen microsomal fractions were untreated (lane 1) or treated with tobacco style extract (lane 2), or with 1.44 Abs280 units (lanes 3, 6, 9 and 12), 0.72 Abs280 units (lanes 4, 7, 10 and 13) or 0.36 Abs280 units (lanes 5, 8, 11 and 14) of partially purified STIL (C18 percolate). Before the dephosphorylation assay was carried out, partially purified STIL (C18 percolate) was pre-treated by microwave-assisted acid hydrolysis (lanes 3-5). Lanes 6-8, salt concentration control. Lanes, 7-10, high temperature control. Lanes, 11-14, sample dilution control. The position of LePRK2 is indicated by an arrow.

Figure 5

STIL promotes in vitro pollen tube germination in tomato. A, Mature pollen was germinated in vitro for 3 h in the presence of increasing amounts of partially purified STIL (C18 percolate). The concentration of STIL is expressed as Abs280 units/μl of Pollen Germination Medium. The Table summarizes the results. SE, standard error; n, number of replicates from independent experiments; *, significant differences relative to water control (*, p < 0.01; **, p < 0.001). B, Pollen tube length fold-increase (left panel) and growth rate (right panel) for different STIL concentrations. The standard error for each average (table in A) was used to calculate the error bars, using error propagation (partial derivatives).