Aminopropyltransferases involved in polyamine biosynthesis localize preferentially in the nucleus of plant cells

Abstract

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.

Figure 1. Histological immunolocalisation of SPDS aminopropyltransferases in Arabidopsis.

Paraffin-embedded sections of different organs of A. thaliana were incubated with anti-SPDS antibodies and an avidin-biotin-peroxidase detection system. Dark brown staining indicates SPDS antibody-specific reaction. Either transverse or oblique sections are shown for: (a) wild-type developed flower, (b) embryo sac, (c) stamen, (d) sepal, (e) wild-type flower primordia, (f) receptacle, (g) gynoecium (i) mature silique, (j, k) embryo, (m, n, o) leaf, (q, r, s) stem, and (u, v, w) root. Specificity of the signal is shown by using preimmune serum as control reactions in (h, l, p, t, x). Abbreviations: C, cotyledon; Ca, cambium; Ch, chloroplast; Co, cortex; E, epidermis; Em, embryo sac; En, peripheral endosperm; G, gynoecium; I, inner integument; N, nucleus; P, pith; Pa, palisade mesophyll; Pe, petal; Ph, phloem; R, radicle; S, embryo; Sc, seed coat; Se, sepal; Si, silique; Sp, stigmatic papillae; St, stamen; Sy, style; Ue, upper epidermis; V, vascular bundle; X, xylem. Bar = 100 µm in (a, e, i, m, q, u); 25 µm in (j, n, r, v); 10 µm in (b, c, d, f, g, h, k, l, o, p, s, t, w, x).

Figure 2. Subcellular localization of aminopropyltransferases as GFP fusion proteins in N.benthamiana.

Translational fusion constructs of aminopropyltransferases to GFP, both at the N-terminus and the C-terminus were transiently expressed in N. benthamiana by agroinfiltration together with a viral nuclear marker fused to mRFP, and analysed with a laser-scanning confocal fluorescence microscope. GFP and mRFP fluorescence spectrum are shown in left and middle column panels. Merged visible and fluoresecent signals are shown in the right column panel. Scale bars: 40 µm.

Figure 3. Localization of aminopropyltransferase-GFP fusion proteins and biochemical fractionation of Arabidopsis transgenic plants.

A, the same constructs in the pMDC83 vector used for transient expression in N.benthamiana were used to obtain Arabidopsis transgenic plants expressing SPDS1, SPDS2 and SPMS as GFP fusion proteins. T2 transgenic plants were selected and used to visualize the GFP fluorescence or the transmitted signal with the laser-scanning confocal microscope. The insets present part of the same areas with higher magnification. Scale bars: 80 µm for the first two column panels, and 40 µm for the next two column panels. At least 10 different seedlings for each construct were analyzed with similar localization pattern. B, SPDS2-GFP transgenic plants were used for biochemical fractionation of soluble cytosol (S) or nuclear enriched pellet fractions (P) and tested by western blotting using anti-SPDS2 affinity purified antibodies, anti-histone H3 antibodies as a nuclear marker, and Ponceau-S staining of the Rubisco protein as a cytosolic marker.

Figure 4. Nuclear localization of SPMS mediated by the presence of SPDS2 in the nucleus of N.benthamiana.

Agroinfiltration studies were performed in N.benthamiana to achieve simultaneous co-expression of GFP-SPMS with either the mRFP fluorescent protein or the translational fusion mRFP-SPDS2 and analysed with a laser-scanning confocal fluorescence microscope. GFP fluorescence spectrum, mRFP fluorescence spectrum, and the visible range are shown for every co-transformation. Scale bars: 40 µm.

Figure 5. BiFC assays between aminopropyltransferases

. Translational fusion constructs of aminopropyltransferase coding sequences to both pYFN43 and pYFC43 were agroinfiltrated into N. benthamiana leaves and tested for fluorescence complementation by laser-scanning confocal microscopy. Scale bars: 40 µm.

Figure 6. Gel filtration analyses of native SPDS aminopropyltransferases.

Total protein extracts from Arabidopsis cell suspensions were size fractionated with two different chromatography columns: HiPrep S300 (A) and Superose 6 (B) and tested for immunoblotting with affinity purified anti-SPDS2 antibodies to estimate the apparent molecular size of native SPDS complexes that resulted approximately the size of a dimer.

Figure 7. Recombinant aminopropyltransferases can associate as homodimers.

Aminopropyltransferase proteins expressed in E. coli as fusion proteins to His-tag were affinity purified with nickel chromatography and size fractionated with a Superdex 200 10/300 GL column. 1.5 mL volume fractions were monitored for absorbance at 280 nm. Two peaks corresponding to the size of monomer and dimer were detectable in every case. The gel filtration chromatography was repeated at least twice for each purified recombinant protein and figure shows a representative elution profile.