Modulation of the polyamine biosynthetic pathway in transgenic rice confers tolerance to drought stress

Abstract

We have generated transgenic rice plants expressing the Datura stramonium adc gene and investigated their response to drought stress. We monitored the steady-state mRNA levels of genes involved in polyamine biosynthesis (Datura adc, rice adc, and rice samdc) and polyamine levels. Wild-type plants responded to the onset of drought stress by increasing endogenous putrescine levels, but this was insufficient to trigger the conversion of putrescine into spermidine and spermine (the agents that are believed to protect plants under stress). In contrast, transgenic plants expressing Datura adc produced much higher levels of putrescine under stress, promoting spermidine and spermine synthesis and ultimately protecting the plants from drought. We demonstrate clearly that the manipulation of polyamine biosynthesis in plants can produce drought-tolerant germplasm, and we propose a model consistent with the role of polyamines in the protection of plants against abiotic stress.

Fig. 1.

Molecular and biochemical characterization of transgenic plants carrying the Dadc transgene. (A) Gel blot analysis of KpnI-digested genomic rice DNA. 84, 81, and 86 represent independent transgenic lines; wt, wild type. (B) Gel blot of total leaf RNA from wild-type and independent lines containing Ubi:Dadc. Blots in A and B were probed with a 0.8-kb DIG-labeled PCR product from Ubi:Dadc. (C) Ethidium bromide gel demonstrating equal loading of total RNA. (D) Polyamine content. Values are means ± SE for control lines (n = 36) and means ± SE in transgenic lines (n = 3). Significance of data for putrescine and spermidine is as follows: ^***, P < 0.001; ^**, 0.01 > P > 0.001; ^*, 0.05 > P > 0.01; remaining values were not significantly different from wild type at P > 0.05.

Fig. 2.

Molecular and biochemical characterization of R₁ progeny of clone 84. (A) Gel blot analysis of total RNA from leaf tissue. wt, wild type. Numbers represent different R₁ progeny from plant 84. A 0.8-kb DIG-labeled PCR probe from Datura adc cDNA was used. (B)UV fluorescence of an ethidium bromide-stained gel showing equal loading of total RNA from plants used in the hybridizations shown in A.(C) Cellular putrescine content. Significance of data for putrescine is as follows: ^*, 0.05 > P > 0.01; remaining values are not significantly different from wild type at P > 0.05.

Fig. 3.

Molecular and biochemical characterization of wild-type and transgenic plants under drought stress. (A) Normalization of rice adc mRNA hybridization signals and quantitation as described in Materials and Methods. (B) Normalization of Datura adc mRNA hybridization signals and quantitation as described in Materials and Methods. wt, wild type (progeny from plant 84-2). The 400-bp and 800-bp DIG-labeled PCR probes from rice and Datura adc cDNAs were used. Each blot represents a typical result of two independent experiments. (C) Membrane was reprobed with the 28S rRNA probe to demonstrate equal loadings. (D) Putrescine content. Values are means ± SE for control lines (n = 12) and means ± SE for transgenic lines (n = 3). Significance of data for putrescine among plants of the same line is designated by ^*, and significance between transgenic lines and wild-type plants at the same time period is designated by #. Significance of data for putrescine is as follows: ^***, P < 0.001; ^**, 0.01 > P > 0.001; ^*, 0.05 > P > 0.01; remaining values were not significantly different from wild type at P > 0.05.

Fig. 4.

Molecular and biochemical characterization of wild-type and transgenic plants under drought stress. (A) Normalization of rice samdc mRNA hybridization signals and quantitation as described in Materials and Methods. Each blot represents a typical result of two independent experiments. (B) Membrane was reprobed with the 28S rRNA probe to determine equal loadings. Spermidine (C) and spermine (D) levels in wild-type and transgenic plants after different periods of drought stress are shown. Values are means ± SE for wild type (n = 12) and means ± SE in transgenic lines (n = 3). Significance of data among the same line is designated by ^*, and significance between transgenic lines and wild-type controls at the same time period is designated by #. Significance of data for spermidine and spermine is as follows: ^**, 0.01 > P > 0.001; ^*, 0.05 > P > 0.01; remaining values were not significantly different from wild type at P > 0.05.

Fig. 5.

Response of rice plants to drought stress. (A) Phenotype of 2-month-old wild-type (WT) and transgenic plants (84-2 and 84-9 lineages) growing in soil after drought stress (6 days). (B) Close-up of rice leaves (wild type on the left and 84-2 on the right).

Fig. 6.

Unified model explaining the role of polyamines in the response of wild-type and transgenic plants expressing Dadc under abiotic stress conditions. The model is considered in more detail in Discussion. Histograms show relative polyamine levels measured in each type of plant. Once putrescine levels exceed the threshold shown, the synthesis of spermidine and spermine is triggered, resulting in protection against drought and other forms of stress.