Polyamines in the life of Arabidopsis: profiling the expression of S-adenosylmethionine decarboxylase (SAMDC) gene family during its life cycle

Abstract

Background: Arabidopsis has 5 paralogs of the S-adenosylmethionine decarboxylase (SAMDC) gene. Neither their specific role in development nor the role of positive/purifying selection in genetic divergence of this gene family is known. While some data are available on organ-specific expression of AtSAMDC1, AtSAMDC2, AtSAMDC3 and AtSAMDC4, not much is known about their promoters including AtSAMDC5, which is believed to be non-functional.

Results: (1) Phylogenetic analysis of the five AtSAMDC genes shows similar divergence pattern for promoters and coding sequences (CDSs), whereas, genetic divergence of 5'UTRs and 3'UTRs was independent of the promoters and CDSs; (2) while AtSAMDC1 and AtSAMDC4 promoters exhibit high activity (constitutive in the former), promoter activities of AtSAMDC2, AtSAMDC3 and AtSAMDC5 are moderate to low in seedlings (depending upon translational or transcriptional fusions), and are localized mainly in the vascular tissues and reproductive organs in mature plants; (3) based on promoter activity, it appears that AtSAMDC5 is both transcriptionally and translationally active, but based on it's coding sequence it seems to produce a non-functional protein; (4) though 5'-UTR based regulation of AtSAMDC expression through upstream open reading frames (uORFs) in the 5'UTR is well known, no such uORFs are present in AtSAMDC4 and AtSAMDC5; (5) the promoter regions of all five AtSAMDC genes contain common stress-responsive elements and hormone-responsive elements; (6) at the organ level, the activity of AtSAMDC enzyme does not correlate with the expression of specific AtSAMDC genes or with the contents of spermidine and spermine.

Conclusions: Differential roles of positive/purifying selection were observed in genetic divergence of the AtSAMDC gene family. All tissues express one or more AtSAMDC gene with significant redundancy, and concurrently, there is cell/tissue-specificity of gene expression, particularly in mature organs. This study provides valuable information about AtSAMDC promoters, which could be useful in future manipulation of crop plants for nutritive purposes, stress tolerance or bioenergy needs. The AtSAMDC1 core promoter might serve the need of a strong constitutive promoter, and its high expression in the gametophytic cells could be exploited, where strong male/female gametophyte-specific expression is desired; e.g. in transgenic modification of crop varieties.

Keywords: 3’UTR; 5’UTR; Arabidopsis; Gene family evolution; Metabolism; Polyamines; Promoter; SAMDC.

Fig. 1

Polyamine biosynthetic pathway. Pathway for the biosynthesis of polyamines in Arabidopsis thaliana; abbreviations: ODC, ornithine decarboxylase (absent in Arabidopsis); ADC, Arginine decarboxylase; SAMDC, S-adenosylmethionine decarboxylase; SPDS, spermidine synthase; SPMS, spermine synthase; tSPMS, thermospermine synthase

Fig. 2

Phylogenetic analyses of the SAMDC genes in Arabidopsis thaliana. a CDS; b upstream elements (UE: promoter); c 5’UTRs; and d 3’UTRs. Evolutionary analyses of the SAMDC sequences was inferred by using the Maximum Likelihood method based on the Tamura-Nei model with boot strapping. The tree with the highest log likelihood is presented here for each phylogenetic analysis. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. Boot strapping with 500 replications was conducted as a test of phylogeny. The percentage of trees in which the associated taxa clustered together is shown next to the branches defined as percent boot strap values. The tree was drawn to scale, with branch lengths measured as number of nucleotide substitutions per site given in the scale bar

Fig. 3

Different constructs of Arabidopsis SAMDC genes. AtSAMDC1, AtSAMDC2, AtSAMDC3, AtSAMDC4, and AtSAMDC5 GUS fusions used for expression analyses of the reporter gene (+1 denotes transcription start site)

Fig. 4

Activity of GUS at early germination stages (24 h and 48 h) in different AtSAMDC constructs. AtSAMDC1 (A1-A4), AtSAMDC2 (B1-B4), AtSAMDC3 (C1-C4, D1-D4), AtSAMDC4 (E1-E4, F1-F4) and AtSAMDC5 (G1-G4, H1-H4) Arabidopsis seeds. The name of individual construct is at the left of individual horizontal panel

Fig. 5

Activity of GUS in 9-day old seedlings. AtSAMDC1 (A1-A2), AtSAMDC2 (B1-B3), AtSAMDC3 (C1-C4), AtSAMDC4 (D1-D4), and AtSAMDC5 (E1-E3)

Fig. 6

Activity of GUS in roots and rosette leaves of 5-week old transgenic plants. a AtSAMDC1 and AtSAMDC2; b AtSAMDC3; c AtSAMDC4;  d AtSAMDC5. Roots (a: a_1–5, b: a_1–4, c: a_1–4, d: a_1–3); and rosette leaves (a: b_1–5, b: b_1–4, c: b_1–4, d: b_1–3)

Fig. 7

Activity of GUS in reproductive parts of mature transgenic plants. a AtSAMDC1 and AtSAMDC2; b AtSAMDC3; c AtSAMDC4; d AtSAMDC5. Flower (a: a_1–5, b: a_1–4, c: a_1–4, d: a_1–3); close up view of anther sac and pollens (a: b_1–5, b: b_1–4, c: b_1–4, d: b_1–3); and embryo (a: c_1–5, b: c_1–4, c: c_1–4, d: c_1–3)

Fig. 8

Gene expression, enzyme assay, and polyamine contents in wild type Arabidopsis thaliana. a relative expression of the five AtSAMDC genes (white bars = 2-day and gray bars = 10-day old seedlings; b AtSAMDC gene expressions in 5-week old mature plant parts; c SAMDC enzyme activity in 5-week old mature plant parts; and (d) polyamine contents in seedlings and mature plant parts (abbreviations used: RL = rosette leaf, CL = cauline leaf, 2 d = 2 day whole seedlings, 10 d = 10 day whole seedlings, wk. = week, RT = root, F = flower, B = bud, LS = lower stem, US = upper stem, GSi = green silique, MSi = mature silique). Data are mean ± SE of 3–4 biological replicates