Phosphate Deficiency Induces the Jasmonate Pathway and Enhances Resistance to Insect Herbivory

Abstract

During their life cycle, plants are typically confronted by simultaneous biotic and abiotic stresses. Low inorganic phosphate (Pi) is one of the most common nutrient deficiencies limiting plant growth in natural and agricultural ecosystems, while insect herbivory accounts for major losses in plant productivity and impacts ecological and evolutionary changes in plant populations. Here, we report that plants experiencing Pi deficiency induce the jasmonic acid (JA) pathway and enhance their defense against insect herbivory. Pi-deficient Arabidopsis (Arabidopsis thaliana) showed enhanced synthesis of JA and the bioactive conjugate JA-isoleucine, as well as activation of the JA signaling pathway, in both shoots and roots of wild-type plants and in shoots of the Pi-deficient mutant pho1 The kinetics of the induction of the JA signaling pathway by Pi deficiency was influenced by PHOSPHATE STARVATION RESPONSE1, the main transcription factor regulating the expression of Pi starvation-induced genes. Phenotypes of the pho1 mutant typically associated with Pi deficiency, such as high shoot anthocyanin levels and poor shoot growth, were significantly attenuated by blocking the JA biosynthesis or signaling pathway. Wounded pho1 leaves hyperaccumulated JA/JA-isoleucine in comparison with the wild type. The pho1 mutant also showed an increased resistance against the generalist herbivore Spodoptera littoralis that was attenuated in JA biosynthesis and signaling mutants. Pi deficiency also triggered increased resistance to S. littoralis in wild-type Arabidopsis as well as tomato (Solanum lycopersicum) and Nicotiana benthamiana, revealing that the link between Pi deficiency and enhanced herbivory resistance is conserved in a diversity of plants, including crops.

Figure 1.

Genes involved in JA biosynthesis and signaling are induced in shoots and roots of Pi-deficient plants. A to I, Expression levels of JAZ10 (A–C), VSP2 (D–F), and LOX2 (G–I) measured in shoots (A, D, and G), whole roots (B, E, and H), and root tips (3 mm; C, F, and I) in Col-0 or the aos and coi1-34 mutants. Plants were grown for 12 d in medium containing 1 mm (black bars) or 100 µm (gray bars) Pi. J, Expression levels of JAZ10, VSP2, and LOX2 measured in shoots of Col-0 (black bars) and pho1-7 (gray bars) grown for 4 weeks in fertilized soil. For A to I, data are means ± sd of three samples obtained from independent plates, with three technical replicates for each sample and each sample consisting of a pool of tissues isolated from 10 plants for shoots, approximately 50 plants for roots, and approximately 150 plants for root tips. For J, data are means ± sd of three samples from plants grown in independent pots and three technical replicates for each sample, with each sample being a pool of three plants. Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; and ***, P < 0.001) according to Student’s t test. Experiments were performed independently three times with similar results.

Figure 2.

Enhanced levels of JA and JA-Ile in Pi-deficient tissues. A, B, E, and F, JA (A and E) and JA-Ile (B and F) contents in leaves (A and B) and roots (E and F) from plants grown for 12 d in medium containing 1 mm or 100 µm Pi. C and D, JA (C) and JA-Ile (D) contents in shoots from plants grown for 12 d in medium containing 1 mm or 100 µm Pi and 1 h after wounding. G to J, JA (G and I) and JA-Ile (H and J) in shoots of 4-week-old soil-grown Col-0, pho1-7, and pho1-2 plants sampled before wounding (G and H) or 1 h after wounding (I and J). For A to F, data are means ± sd of five samples obtained from independent plates, with each sample consisting of a pool of tissues isolated from 20 plants for leaves and 100 plants for roots. For G to J, data are means ± sd of five samples from plants grown in independent pots, with each sample being a pool of three plants. Asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; and ***, P < 0.001) according to Student’s t test. For G to J, statistical analysis was performed comparing Col-0 with each of the pho1 mutants. Experiments were performed independently one time (I and J), two times (C, D, G, and H), or three times (A, B, E, and F) with similar results. FW, Fresh weight.

Figure 3.

The kinetics of JAZ10 induction by Pi deficiency is influenced by PHR1. JAZ10 (A) and IPS1 (B) expression is shown in Col-0 and phr1-1 seedlings grown for 7 or 10 d in medium containing 1 mm (gray bars) or 20 µm (black bars) Pi. Data are means ± sd of three samples obtained from independent plates, with three technical replicates for each sample and each sample consisting of a pool of tissues isolated from 20 seedlings. Asterisks denote statistical significance (**, P < 0.01 and ***, P < 0.001) according to Student’s t test. Statistical analysis was done comparing Col-0 and phr1-1 within each treatment. Experiments were performed independently three times with similar results.

Figure 4.

Contribution of the JA pathway to the phenotype of Pi deficiency. A to E, Wild-type and mutant plants grown in fertilized soil for 4 weeks were compared for overall appearance (A), shoot fresh weight (B), shoot anthocyanin content (C), and the expression of the IPS1 (D) and MGD3 (E) genes in shoots. In A, the pots containing Col-0 and pho1-7 plants at left are the same as those shown at right. F and G, JA (F) and Ja-Ile (G) contents in rosette leaves from plants grown for 4 weeks in soil. H, The response of the primary root to low Pi was assessed by growing plants on agar-solidified medium containing 1 mm Pi for 7 d before transferring them to similar medium containing either 1 mm (gray bars) or 10 µm (black bars) Pi for 3 d. Growth of the primary root in the last 3 d was measured. I, Anthocyanin levels in shoots of plants grown for 7 d in agar-solidified medium containing 1 mm Pi followed by transfer to medium containing either 1 mm (gray bars) or 10 µm (black bars) Pi for 5 d. J, Fresh weight of the rosettes of Col-0, aos, and coi1-34 plants grown for 18 d in agar-solidified medium containing 1 mm, 100 µm, or 20 µm Pi. For B, data are means ± sd of 20 plants grown in individual pots. For C, data are means ± sd of five plants grown in independent pots. For D and E, data are means ± sd of three samples from plants grown in independent pots and three technical replicates for each sample, with each sample being a pool of three plants. For F and G, data are means ± sd of five samples from plants grown in independent pots, with each sample being an individual plant for Col-0, coi1-1, and aos and a pool of three plants for pho1-7, pho1-7 coi1-1, and pho1-7 aos. For H, data are means ± sd of 30 individual roots from three independent plates. For I and J, data are means ± sd of 20 to 30 plants grown in five independent plates. For B to G, values marked with lowercase letters were statistically significantly different from those for other groups marked with different letters (P < 0.05, ANOVA with the Tukey-Kramer honestly significant difference [HSD] test). n.d., Not detectable. For H to J, statistical analysis was done comparing Col-0 with the coi1-34 and aos mutants within each treatment, and asterisks denote statistical significance (*, P < 0.05 and **, P < 0.01) according to Student’s t test. Experiments were performed independently one time (F, G, and J), two times (B, H, and I), or three times (C–E) with similar results. FW, Fresh weight.

Figure 5.

The pho1 mutant is resistant to the generalist herbivore S. littoralis but not the specialist herbivore P. brassicae. A and B, Survival rate (A) and larvae weight (B) of S. littoralis caterpillars after 12 d of feeding on 4-week-old plants grown in fertilized soil. C and D, Survival rate (C) and larvae weight (D) of P. brassicae caterpillars after 7 d of feeding on 4-week-old plants grown in fertilized soil. E, Representative images of Col-0 and pho1-7 plants after feeding on S. littoralis (top) and P. brassicae (bottom). Data are means ± sd of 20 to 30 caterpillars for A and B and 10 to 20 caterpillars for C and D feeding on individually grown plants. Values in A and B marked with lowercase letters were statistically significantly different from those for other groups marked with different letters (P < 0.05, ANOVA with the Tukey-Kramer HSD test). For C and D, Student’s t test was used. Experiments were performed independently two times (C and D) or three times (A and B) with similar results.

Figure 6.

Long-term Pi deficiency induces resistance to S. littoralis herbivory. Plants were grown from seeds on a soilless aeroponic system supplemented with Hoagland nutrient solution containing either 1 mm Pi or no added Pi. Weight (left) and survival rate (right) of S. littoralis caterpillars were measured after either 12 d of feeding on 4-week-old Arabidopsis (A) or 12 d of feeding on 3-week-old tomato (B) or N. benthamiana (C). Data are means ± sd of 10 to 20 caterpillars feeding on individually grown plants. Student’s t test was performed, and asterisks denote statistical significance (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). Experiments were performed independently two times (B and C) or three times (A) with similar results.