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SAM levels, gene expression of SAM synthetase, methionine synthase and ACC oxidase, and ethylene emission from N. suaveolens flowers.

Roeder S, Dreschler K, Wirtz M, Cristescu SM, van Harren FJ, Hell R, Piechulla B - Plant Mol. Biol. (2009)

Bottom Line: The SAM concentrations in flowers of Nicotiana suaveolens were determined during day/night cycles and found to fluctuate rhythmically between 10 and 50 nmol g(-1) fresh weight.Ethylene, which is synthesized from SAM, reached only low levels of 1-2 ppbv in N. suaveolens flowers.It is emitted in a burst at the end of the life span of the flowers, which correlates with the increased expression of the 1-aminocyclopropane-1-carboxylate oxidase (NsACO).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Institute of Biological Sciences, University of Rostock, Albert-Einstein-Strasse 3, Rostock, Germany.

ABSTRACT
S'adenosyl-L: -methionine (SAM) is a ubiquitous methyl donor and a precursor in the biosynthesis of ethylene, polyamines, biotin, and nicotianamine in plants. Only limited information is available regarding its synthesis (SAM cycle) and its concentrations in plant tissues. The SAM concentrations in flowers of Nicotiana suaveolens were determined during day/night cycles and found to fluctuate rhythmically between 10 and 50 nmol g(-1) fresh weight. Troughs of SAM levels were measured in the evening and night, which corresponds to the time when the major floral scent compound, methyl benzoate, is synthesized by a SAM dependent methyltransferase (NsBSMT) and when this enzyme possesses its highest activity. The SAM synthetase (NsSAMS1) and methionine synthase (NsMS1) are enzymes, among others, which are involved in the synthesis and regeneration of SAM. Respective genes were isolated from a N. suaveolens petal cDNA library. Transcript accumulation patterns of both SAM regenerating enzymes matched perfectly those of the bifunctional NsBSMT; maximum mRNA accumulations of NsMS1 and NsSAMS1 were attained in the evening. Ethylene, which is synthesized from SAM, reached only low levels of 1-2 ppbv in N. suaveolens flowers. It is emitted in a burst at the end of the life span of the flowers, which correlates with the increased expression of the 1-aminocyclopropane-1-carboxylate oxidase (NsACO).

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RNA expression profile of methionine synthase, SAM synthetase and ACC oxidase of N. suaveolens at different times during the day and during flower development. Plants were grown under 16 h light (L) and 8 h darkness (D; 10 pm to 6 am) (details of growing conditions are given in M and M). Petals of N. suaveolens were harvested at indicated time points at six consecutive days after flower opening. RNA was isolated and 5 μg of total RNA was separated on denaturing agarose gels. RNA was blotted and hybridised with NsSAMS1, NsMS1 and NsACO probes (Fig. S4). Blots were rehybridized with 18S rDNA probe to allow normalisation. The quantification was based on two independent experiments and duplicated Northern blots, highest expression levels were set at 100% and relative transcript levels were calculated, SE is indicated (n = 4). (A) methionine synthase; (B) SAM synthetase; (C) ACC oxidase
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Fig4: RNA expression profile of methionine synthase, SAM synthetase and ACC oxidase of N. suaveolens at different times during the day and during flower development. Plants were grown under 16 h light (L) and 8 h darkness (D; 10 pm to 6 am) (details of growing conditions are given in M and M). Petals of N. suaveolens were harvested at indicated time points at six consecutive days after flower opening. RNA was isolated and 5 μg of total RNA was separated on denaturing agarose gels. RNA was blotted and hybridised with NsSAMS1, NsMS1 and NsACO probes (Fig. S4). Blots were rehybridized with 18S rDNA probe to allow normalisation. The quantification was based on two independent experiments and duplicated Northern blots, highest expression levels were set at 100% and relative transcript levels were calculated, SE is indicated (n = 4). (A) methionine synthase; (B) SAM synthetase; (C) ACC oxidase

Mentions: To determine the gene expression levels at different time points during flower development and at different daily time points, petals were collected in 3 h intervals from the 1st to 6th day after N. suaveolens flower opening and the RNA was isolated and hybridised with the NsMS1, NsSAMS1 and NsACO probes. Examples of the expression of the NsMS1 and NsSAMS1 genes are exemplarily shown in Fig. S4. The quantitative analysis revealed simultaneous oscillations from the 1st to the 6th day of flowering for the NsMS1 and NsSAMS1 (Fig. 4A, B). The expression maxima were in the late afternoon/early evening and possess a typical nocturnal expression pattern. The methionine synthase transcripts reached their highest amplitudes in 2- and 3-day-old flowers and, thereafter, the amplitude decreased slowly during flower senescense. The SAM synthetase mRNA oscillations varied around 60–80%, with the maximum amplitude being determined on day 5, whereas in senescent flowers (day 6), SAM synthetase transcript levels decreased.Fig. 4


SAM levels, gene expression of SAM synthetase, methionine synthase and ACC oxidase, and ethylene emission from N. suaveolens flowers.

Roeder S, Dreschler K, Wirtz M, Cristescu SM, van Harren FJ, Hell R, Piechulla B - Plant Mol. Biol. (2009)

RNA expression profile of methionine synthase, SAM synthetase and ACC oxidase of N. suaveolens at different times during the day and during flower development. Plants were grown under 16 h light (L) and 8 h darkness (D; 10 pm to 6 am) (details of growing conditions are given in M and M). Petals of N. suaveolens were harvested at indicated time points at six consecutive days after flower opening. RNA was isolated and 5 μg of total RNA was separated on denaturing agarose gels. RNA was blotted and hybridised with NsSAMS1, NsMS1 and NsACO probes (Fig. S4). Blots were rehybridized with 18S rDNA probe to allow normalisation. The quantification was based on two independent experiments and duplicated Northern blots, highest expression levels were set at 100% and relative transcript levels were calculated, SE is indicated (n = 4). (A) methionine synthase; (B) SAM synthetase; (C) ACC oxidase
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Related In: Results  -  Collection

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Fig4: RNA expression profile of methionine synthase, SAM synthetase and ACC oxidase of N. suaveolens at different times during the day and during flower development. Plants were grown under 16 h light (L) and 8 h darkness (D; 10 pm to 6 am) (details of growing conditions are given in M and M). Petals of N. suaveolens were harvested at indicated time points at six consecutive days after flower opening. RNA was isolated and 5 μg of total RNA was separated on denaturing agarose gels. RNA was blotted and hybridised with NsSAMS1, NsMS1 and NsACO probes (Fig. S4). Blots were rehybridized with 18S rDNA probe to allow normalisation. The quantification was based on two independent experiments and duplicated Northern blots, highest expression levels were set at 100% and relative transcript levels were calculated, SE is indicated (n = 4). (A) methionine synthase; (B) SAM synthetase; (C) ACC oxidase
Mentions: To determine the gene expression levels at different time points during flower development and at different daily time points, petals were collected in 3 h intervals from the 1st to 6th day after N. suaveolens flower opening and the RNA was isolated and hybridised with the NsMS1, NsSAMS1 and NsACO probes. Examples of the expression of the NsMS1 and NsSAMS1 genes are exemplarily shown in Fig. S4. The quantitative analysis revealed simultaneous oscillations from the 1st to the 6th day of flowering for the NsMS1 and NsSAMS1 (Fig. 4A, B). The expression maxima were in the late afternoon/early evening and possess a typical nocturnal expression pattern. The methionine synthase transcripts reached their highest amplitudes in 2- and 3-day-old flowers and, thereafter, the amplitude decreased slowly during flower senescense. The SAM synthetase mRNA oscillations varied around 60–80%, with the maximum amplitude being determined on day 5, whereas in senescent flowers (day 6), SAM synthetase transcript levels decreased.Fig. 4

Bottom Line: The SAM concentrations in flowers of Nicotiana suaveolens were determined during day/night cycles and found to fluctuate rhythmically between 10 and 50 nmol g(-1) fresh weight.Ethylene, which is synthesized from SAM, reached only low levels of 1-2 ppbv in N. suaveolens flowers.It is emitted in a burst at the end of the life span of the flowers, which correlates with the increased expression of the 1-aminocyclopropane-1-carboxylate oxidase (NsACO).

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Institute of Biological Sciences, University of Rostock, Albert-Einstein-Strasse 3, Rostock, Germany.

ABSTRACT
S'adenosyl-L: -methionine (SAM) is a ubiquitous methyl donor and a precursor in the biosynthesis of ethylene, polyamines, biotin, and nicotianamine in plants. Only limited information is available regarding its synthesis (SAM cycle) and its concentrations in plant tissues. The SAM concentrations in flowers of Nicotiana suaveolens were determined during day/night cycles and found to fluctuate rhythmically between 10 and 50 nmol g(-1) fresh weight. Troughs of SAM levels were measured in the evening and night, which corresponds to the time when the major floral scent compound, methyl benzoate, is synthesized by a SAM dependent methyltransferase (NsBSMT) and when this enzyme possesses its highest activity. The SAM synthetase (NsSAMS1) and methionine synthase (NsMS1) are enzymes, among others, which are involved in the synthesis and regeneration of SAM. Respective genes were isolated from a N. suaveolens petal cDNA library. Transcript accumulation patterns of both SAM regenerating enzymes matched perfectly those of the bifunctional NsBSMT; maximum mRNA accumulations of NsMS1 and NsSAMS1 were attained in the evening. Ethylene, which is synthesized from SAM, reached only low levels of 1-2 ppbv in N. suaveolens flowers. It is emitted in a burst at the end of the life span of the flowers, which correlates with the increased expression of the 1-aminocyclopropane-1-carboxylate oxidase (NsACO).

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