Expression of a heterologous S-adenosylmethionine decarboxylase cDNA in plants demonstrates that changes in S-adenosyl-L-methionine decarboxylase activity determine levels of the higher polyamines spermidine and spermine

Abstract

We posed the question of whether steady-state levels of the higher polyamines spermidine and spermine in plants can be influenced by overexpression of a heterologous cDNA involved in the later steps of the pathway, in the absence of any further manipulation of the two synthases that are also involved in their biosynthesis. Transgenic rice (Oryza sativa) plants engineered with the heterologous Datura stramonium S-adenosylmethionine decarboxylase (samdc) cDNA exhibited accumulation of the transgene steady-state mRNA. Transgene expression did not affect expression of the orthologous samdc gene. Significant increases in SAMDC activity translated to a direct increase in the level of spermidine, but not spermine, in leaves. Seeds recovered from a number of plants exhibited significant increases in spermidine and spermine levels. We demonstrate that overexpression of the D. stramonium samdc cDNA in transgenic rice is sufficient for accumulation of spermidine in leaves and spermidine and spermine in seeds. These findings suggest that increases in enzyme activity in one of the two components of the later parts of the pathway leading to the higher polyamines is sufficient to alter their levels mostly in seeds and, to some extent, in vegetative tissue such as leaves. Implications of our results on the design of rational approaches for the modulation of the polyamine pathway in plants are discussed in the general framework of metabolic pathway engineering.

Figure 1

Generation and molecular characterization of transgenic rice plants expressing the D. stramonium samdc cDNA. A, Map of Ubi::Dsamdc showing transcription unit, relevant restriction sites, and primers used for PCR and RT-PCR analyses. The D. stramonium samdc cDNA is 1.839 kb in size. KpnI has a single restriction site in the plasmid. Nos, Nopaline synthase. Arrows represent primers and length of amplified fragment. B, DNA gel-blot analysis of transgenic rice plants. Genomic DNA (10 μg) was digested with KpnI and probed with the 0.9-kb DIG-labeled PCR product from Ubi::Dsamdc. Exposure time was 10 min; wt, wild type; numbers represent putative transgenic plants; L, molecular size marker (1-kb DNA ladder, Invitrogen, Carlsbad, CA). C, RT-PCR analysis of D. stramonium samdc cDNA (0.9 kb) from total RNA extracted from controls and plants transformed with Ubi::Dsamdc. L, Molecular size marker (1-kb DNA ladder, Invitrogen); +ve, positive control, plasmid Ubi::Dsamdc; −ve, negative control (water); numbers indicate independent transgenic plants; wt, wild type. D, RT-PCR analysis of rice samdc from total RNA extracted from controls and plants transformed with Ubi::Dsamdc. L, Molecular size marker (1-kb DNA ladder, Invitrogen); +ve, positive control, plasmid Ubi::Dsamdc; −ve, negative control (water); numbers represent indicate independent transgenic plants; wt, wild type.

Figure 2

Transcript accumulation in rice leaves. A, Normalization of hybridization signals in leaf tissue after densitometric analysis of autoradiographs. D. stramonium samdc mRNA levels were quantified, and the resulting values were normalized using values obtained from RNA loading levels. Column size represents the relative D. stramonium samdc mRNA level generated by comparing the normalized values of each lane with that of the highest expressing sample. B, Gel-blot analyses of total RNA from transgenic leaf tissue (WT, wild type 2, 3, 4, 17, 34, 45, 72, 75, 81, 98, 105, and 106). A 0.9-kb DIG-labeled PCR probe from D. stramonium samdc cDNA was used. Exposure time was 10 min. C, Gel-blot analyses of total RNA from transgenic leaf tissue (WT, wild type 2, 3, 4, 17, 34, 45, 72, 75, 81, 98, 105, and 106). A 0.7-kb DIG-labeled PCR probe from rice samdc cDNA was used. Exposure time was 20 min. D, Gel-blot analyses of total RNA from transgenic leaf tissue (WT, wild type 2, 3, 4, 17, 34, 45, 72, 75, 81, 98, 105, and 106). A 0.9-kb DIG-labeled PCR probe from rice spd syn cDNA was used. Exposure time was 30 min. E, UV fluorescence of ethidium bromide-stained gel showing equal amount of total RNA loading from plants used for the hybridization shown above.

Figure 3

Biochemical characterization of transgenic rice plants expressing Ubi::Dsamdc. A, SAMDC enzyme activity in different transgenic lines compared with appropriated controls. Values are means ± se for control lines (n = 6) and means ± se in transgenic lines (n = 4). SAMDC activity in clones 4, 72, 81, 98, and 105 was significantly different from controls at P < 0.01; for clone 3 at P < 0.001. Remaining values were not significantly different from control levels at P > 0.05. B, Cellular polyamine levels in controls and 12 representative transgenic plants. Values are means ± se in control lines (n = 36) and means ± se in transgenic lines (n = 9). Putrescine levels were significantly different from controls at P < 0.01 for clone 98 and P < 0.05 for clone 105. Spermidine levels were significantly different from controls at P < 0.05 for all clones. Spermine levels were not significantly different from controls at P > 0.05.

Figure 4

Rice ADC, ODC, DAO, and PAO activities in leaf tissue. Values are means ± se in control lines (n = 4) and means ± se in transgenic lines (n = 4). ADC activity was significantly different from control at P < 0.01 for clone 98 and at P < 0.05 for clone 105. ODC activity was significantly different from control at P < 0.01 for clones 98 and 105. ADC, ODC, DAO, and PAO activities were not significantly different from controls at P > 0.05 in any of the remaining lines.

Figure 5

Polyamine levels in controls and Ubi::Dsamdc-containing seeds. Values are means ± se in control lines (n = 36) and means ± se in transgenic lines (n = 3). Putrescine levels were significantly different from controls at P < 0.05 for clones 98 and 106. Spermidine levels were significantly different from controls at P < 0.01 and P < 0.05 for clones 98 and 106, respectively. Spermine levels were significantly different from control at P < 0.05 for clones 98 and 106. Remaining values were not significantly different from control levels at P > 0.05.