DNA is taken up by root hairs and pollen, and stimulates root and pollen tube growth

Abstract

Phosphorus (P) enters roots as inorganic phosphate (P(i)) derived from organic and inorganic P compounds in the soil. Nucleic acids can support plant growth as the sole source of P in axenic culture but are thought to be converted into P(i) by plant-derived nucleases and phosphatases prior to uptake. Here, we show that a nuclease-resistant analog of DNA is taken up by plant cells. Fluorescently labeled S-DNA of 25 bp, which is protected against enzymatic breakdown by its phosphorothioate backbone, was taken up and detected in root cells including root hairs and pollen tubes. These results indicate that current views of plant P acquisition may have to be revised to include uptake of DNA into cells. We further show that addition of DNA to P(i)-containing growth medium enhanced the growth of lateral roots and root hairs even though plants were P replete and had similar biomass as plants supplied with P(i) only. Exogenously supplied DNA increased length growth of pollen tubes, which were studied because they have similar elongated and polarized growth as root hairs. Our results indicate that DNA is not only taken up and used as a P source by plants, but ironically and independent of P(i) supply, DNA also induces morphological changes in roots similar to those observed with P limitation. This study provides, to our knowledge, first evidence that exogenous DNA could act nonspecifically as signaling molecules for root development.

Figure 1.

Uptake of externally supplied DNA by Arabidopsis roots. Plants were grown on 0.5× MS medium for 11 d before adding 1 μm Cy3-labeled S-DNA (B and E) or 1 μm free Cy3 (H) or 1 μm rhodamine-labeled dextran (K) to the plates and incubated for 3 h. D is an enlarged image of a living root hair cell as indicated by its cytoplasmic streaming (Supplemental Video S1). No fluorescence was observed in plants incubated with free Cy3 (H) or rhodamine-labeled dextran (K). Living root cells were visualized after incubation with 5 μg mL⁻¹ FDA for 1 min (C, F, I, and L). A, D, G, and J are bright-field images of fluorescence images B and C, E and F, H and I, and K and L, respectively. Small square in K shows a fluorescence image of rhodamine-labeled dextran. Images were taken with a confocal microscope.

Figure 2.

Dry weight, P content, and root growth of Arabidopsis plants grown on medium with and without DNA. Dry weight (A), P content (B), and primary root length (F) were similar in plants grown with and without DNA while number of lateral root (C), lateral root length (D), and root hair length (E) increased in response to addition of DNA. Seeds were germinated and cultivated for 14 d (A and B) or 11 d (C–F) on medium containing adequate supply of Pi (5.7 μg KH₂PO₄ per mL growth medium) without DNA (−DNA) or with DNA (+DNA, 0.8 mg mL⁻¹) added. Bars of A and B represent average and sem of four plates with 100 Arabidopsis plants per plate. Bars represent averages and sem of five plants (C, D, and F) or 10 roots hairs on each of three plants (E). Different letters indicate significant differences at P < 0.005 (C and D) or P < 0.0001 (E). Error bars indicate sem.

Figure 3.

Root growth of axenic Arabidopsis plants on medium with or without externally supplied DNA. Plants were grown for 11 d on medium containing an adequate concentration of Pi (5.7 μg KH₂PO₄ per mL growth medium without DNA [A, C, and E] or with DNA [0.8 mg DNA per mL growth medium] added [B, D, and F]). Root hair length (D and F) and number of lateral root (B) increased in response to the addition of DNA. C and D are enlarged images of a and b, respectively. E and F are enlarged images of c and d, respectively. Bright-field images of E and F were taken with a confocal microscope.

Figure 4.

Uptake of Cy3-labeled S-DNA and rhodamine-labeled dextran by Arabidopsis pollen tubes. Bright-field images are shown (A, D, and G) for pollen grown on germination medium for 5 h and incubated with 1 μm Cy3-S-DNA (B), 1 μm Cy3 dye (E), and 1 μm rhodamine dextran (H), respectively, for 1 h. Cy3 (E) and rhodamine dextran (H) were not taken up by pollen. Living root cell were visualized by staining with 5 μg mL⁻¹ FDA for 1 min (C, F, and I). Images were viewed with a confocal microscope.

Figure 5.

Pollen tube growth in response to external DNA supply. Pollen tubes of Arabidopsis (A) and N. benthamiana (B) grow longer with DNA (0.4 mg plasmid per mL germination buffer) added to the growth medium. The length of Arabidopsis (C) and N. benthamiana (D) pollen tubes was significantly (P < 0.0001) greater in DNA-containing medium as indicated by different letters. Pollen was viewed after 3 h of incubation in germination medium. Different letters indicate significant differences at P < 0.0001 (C–D). Error bars indicate sem.