| Abstract |
The polyamines (PAs) spermidine (Spd) and spermine (Spm) and their obligatory precursor, putrescine (Put), are small aliphatic amines, ubiquitous in living organisms. Even though their exact role and function is not yet understood, many studies relate them with a wide range of physiological phenomena, from cellular development and differentiation to stress-induced responses. The preliminary and determinant stage of polyamine biosynthesis is the decarboxylation of ornithine and arginine from the homonymous decarboxylases, to putrescine and agmatine, respectively. Their homeostasis in plant cells is regulated by the rate of their biosynthesis, conjugation and catabolism. In the present study, the homeostasis of polyamines was examined, in an easily regenetated species, the tobacco (Nicotiana tabacum L) and in a woody perennial plant species, the grapevine (Vitis vinifera L.), which does not easily respond to in vitro morphogenic manipulations. Detailed spatial and temporal profiles of the endogenous soluble (S-), soluble-conjugated (SH-) and insoluble-conjugated (PH-) fractions of Put, Spd and Spm are shown in nearly all plant organs of both plant species. The profile of expression of enzymes of PA biosynthesis and catabolism at the transcript, protein and specific activity levels along with the endogenous concentrations of the precursor amino acids arginine and ornithine are also presented. The results reveal that PA titers and biosynthesis follow a basipetal decrease along the tobacco plant axis; the transcripts of the genes encoding the arginine decarboxylase (ADC), S-adenosyl-L-methionine decarboxylase (SAMDC) and spermidine synthase (SPDS) enzymes and ADC protein do not coincide with enzyme activities in the youngest tissues; Spd synthesis strongly correlates with intense cell divisions; the S-/tot-PA ratios gradually increase basipetally, whereas the conjugated PAs decrease with Spd to mainly determine these changes; total leaf size negatively correlates with PA titers and synthesis; PA biosynthetic enzymes are down-regulated during development; the titers of Arg and Orn and arginase activity increase with age; the shoot apical meristem is the main site of Spd and Spm biosynthesis, whereas the root synthesizes mostly Put. Analysis of Nicotiana tabacum plants also reveals temporally and spatially discrete patterns of di- and polyamine oxidases (DAO and PAO), peroxidases (POXs) and putrescine N-methyltransferase (PMT) expression in shoot apical cells, leaves (apical, peripheral and central regions), acropetal and basipetal petiole regions, internodes, young and old roots. Enzyme activity assays show that DAO preferentially oxidizes the aliphatic diamine putrescine, whereas PAO oxidizes the triamine spermidine and, preferentially, the tetramine spermine. Both DAO and PAO are known to produce hydrogen peroxide, a recognized plant signal molecule and substrate for POXs. Expression analyses reveal that amine oxidases in developing tobacco tissues precede and overlap with POXs and lignification and therefore could be a good marker for vascular tissue development. In mature and old tobacco tissues expression of amine oxidase and POX activities also coincide with putrescine N-methyltransferase (PMT), whereas in young roots PMT dramatically increases, independently of amine oxidases. In both hypergeous and hypogeous tissues, however, DAO and PAO expression occurs in cells destined to undergo lignification, suggesting a different in situ localization of amine oxidases, POXs and PMT. Αll fractions of Spd and Spm decrease during development in both tobacco and grapevine. In grapevine, however, in contrast to tobacco, the three Put fractions increase with increasing leaf age. Total PAs are almost 5-fold higher in the youngest leaf of tobacco than in grapevine. In middle age, fully developed leaves, which are mostly used in plant tissue culture techniques and for isolation of protoplasts, tot-Put is significantly higher, and S-Put is 5-fold higher in grapevine, whereas tot-Spd and tot-Spm are significantly greater in tobacco. Among organogenic and non-organogenic grapevine apical leaves, laminas and petiolar stubs regions differ significantly in their PA levels. Apical petiolar stubs regions capable of developing adventitious shoots could be distinguished by their lower Put and higher free Spd and free Spm contents from apical petiolar stubs regions incapable of forming adventitious shoots. However, in laminas regions (which are not organogenic) of organogenic leaves significantly lower Put, free Spd and free Spm levels are present. The results suggest a free PA transport towards organogenic sites, during the organogenic procedure.
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