Titanium nanoparticles activate a transcriptional response in Arabidopsis that enhances tolerance to low phosphate, osmotic stress and pathogen infection

Abstract

Titanium is a ubiquitous element with a wide variety of beneficial effects in plants, including enhanced nutrient uptake and resistance to pathogens and abiotic stresses. While there is numerous evidence supporting the beneficial effects that Ti fertilization give to plants, there is little information on which genetic signaling pathways the Ti application activate in plant tissues. In this study, we utilize RNA-seq and ionomics technologies to unravel the molecular signals that Arabidopsis plants unleash when treated with Ti. RNA-seq analysis showed that Ti activates abscisic acid and salicylic acid signaling pathways and the expression of NUCLEOTIDE BINDING SITE-LEUCINE RICH REPEAT receptors likely by acting as a chemical priming molecule. This activation results in enhanced resistance to drought, high salinity, and infection with Botrytis cinerea in Arabidopsis. Ti also grants an enhanced nutritional state, even at suboptimal phosphate concentrations by upregulating the expression of multiple nutrient and membrane transporters and by modifying or increasing the production root exudates. Our results suggest that Ti might act similarly to the beneficial element Silicon in other plant species.

Keywords: abiotic stress; beneficial elements; ionomic; nutrient starvation; phytohormones; silicon; titanium oxide; transcriptomic.

Figure 1

Effect of Titanium (Ti) on Arabidopsis growth and inorganic phosphate (Pi) content. (A) Primary root length and (B) shoot fresh weight were determined ten days after treatment with 0.2, 0.25, 0.5, 1, 2, 4, and 8 mM Ti. Concentration of free Pi was also determined in roots (C) and shoots (D) from experiments in (A, B) No added Ti (0) was used as control treatment. Different letters indicate statistically significant differences according to Tukey’s significance test (p< 0.05).

Figure 2

Effect of Titanium (Ti) on Arabidopsis inorganic phosphate (Pi) content under variable Pi conditions. Arabidopsis seedlings were grown under 0.005, 0.05, 0.125, 0.5, and 1 mM Pi and treated with Ti 2 mM. Free Pi content was determined in root (A) and shoot (B) after ten days of germination. No added Ti was used as control treatment. Different letters indicate statistically significant differences according to Tukey’s significance test (p< 0.05).

Figure 3

Effect of Titanium (Ti) on elemental content of Arabidopsis. Content (mg of the analyte/kg of the sample) of 19 elements was determined in shoot and root three days (A, B) and 10 days (C, D) after the treatment with Ti 2 mM. Only elements with statistically significant changes according to Tukey’s significance test (p< 0.05) are presented. Changes are presented as percentage changes.

Figure 4

Transcriptomic responses and enriched Gene Ontology (GO) categories of differentially expressed genes (DEGs) in response to Titanium (Ti) in Arabidopsis. (A) Shared upregulated DEGs between roots and shoots. Top 30 enriched GO categories of upregulated DEGs in roots (B) and shoots (C) in response to Ti treatment.

Figure 5

Effect of Titanium (Ti) on expression of nutrient acquisition related genes. Expression level (mean log2F.C.) of genes encoding transporters of (A) macro and (B) microelements, and other related molecules (C). Gray squares indicate that the gene was filtered-out in the filtering step of the edgeR pipeline due to low CPMs. All heatmaps share the same color scale.

Figure 6

Effect of Titanium (Ti) on the expression of abscisic acid (ABA) and drought tolerance related genes in Arabidopsis. (A) Mean normalized expression of ABA related genes. Gray squares indicate that the gene was filtered-out in the filtering step of the edgeR pipeline due to low CPMs. (B) Percentage of surviving plants seven days after rewatering drought-stressed plants (n=60). (C) Representative photographs of plants exposed to drought before re-watering, and one hour and one week after rewatering.

Figure 7

Effect of Titanium (Ti) on Arabidopsis tolerance to high salinity. (A) Percentage of surviving plants seven days after exposure to 80 mM NaCl. Plants were germinated in MS and then transferred to MS containing 80 mM NaCl (blue bars). Plants treated with Ti were germinated in MS medium containing 2 mM Ti and then transferred to MS media with 80 mM NaCl (green bars) or MS media containing 80 mM NaCl and 2 mM Ti (yellow bars). (B) Representative photographs of plants under each treatment seven days after salt-stress exposure. Relative expression of salt-stress related genes in Arabidopsis in roots: PYL4 (C), ABI5 (D), AAO3 (E) one day after Ti treatment; and PYL4 (F), ABI5 (G), and AAO3 (H). Different letters indicate statistically significant differences according to Tukey’s significance test (p< 0.05).

Figure 8

Effect of Titanium (Ti) on the expression of pathogen responsive genes and on Arabidopsis-Botrytis cinerea interaction. (A) Mean normalized expression levels of NBS-LRR receptors genes. (B) Mean normalized expression levels of salicylic acid (SA) and systemic acquired response related genes. Numbers bellow the heatmap indicate the function of the genes; 1) SA regulatory modules, 2) SA biosynthesis positive regulators, 3) SA biosynthesis negative regulators, 4) SA biosynthesis, 5) SA receptors, 6) SA responsive genes, and 7) cell death. (C) Representative photographs of plants under each treatment at one, three, and seven days after B. cinerea infection. Each segment of a bar are equal to 2.5 mm. Individual leaves were cropped from the original photograph to make the composition; no further modifications or picture enhancement were made. (D) Area of the lesion (mm²) in B. cinerea infected plants. Treatments with the same letter are not significantly different. n.s.: not significant. Different letters indicate statistically significant differences according to Tukey’s significance test (p< 0.05). (E) Percentage of leaves with necrotic spots. Only p-values of the two-tail chi-square test are presented when the values are statistically significant. In (A) and (B); gray squares indicate that the gene was filtered-out in the filtering step of the edgeR pipeline due to low CPMs. Both heatmaps share the same color scale. Both (D) and (E), share the same color key.