Sustained defense response via volatile signaling and its epigenetic transcriptional regulation

Abstract

Plants perceive volatiles emitted from herbivore-damaged neighboring plants to urgently adapt or prime their defense responses to prepare for forthcoming herbivores. Mechanistically, these volatiles can induce epigenetic regulation based on histone modifications that alter the transcriptional status of defense genes, but little is known about the underlying mechanisms. To understand the roles of such epigenetic regulation of plant volatile signaling, we explored the response of Arabidopsis (Arabidopsis thaliana) plants to the volatile β-ocimene. Defense traits of Arabidopsis plants toward larvae of Spodoptera litura were induced in response to β-ocimene, through enriched histone acetylation and elevated transcriptional levels of defense gene regulators, including ethylene response factor genes (ERF8 and ERF104) in leaves. The enhanced defense ability of the plants was maintained for 5 d but not over 10 d after exposure to β-ocimene, and this coincided with elevated expression of those ERFs in their leaves. An array of histone acetyltransferases, including HAC1, HAC5, and HAM1, were responsible for the induction and maintenance of the anti-herbivore property. HDA6, a histone deacetylase, played a role in the reverse histone remodeling. Collectively, our findings illuminate the role of epigenetic regulation in plant volatile signaling.

Figure 1

Defensive properties of Arabidopsis plant accessions in response to β-ocimene. Col-0, Ty-0, and Kas-1 plants were exposed to the headspace volatiles released from TEC with (+) or without (−) 200 µg of β-ocimene during 26–32 dap. A S. litura larva was released onto the plant at 32 dap and the net body weight of the larva gained during 4 d was determined. Data represent the mean and standard error (n = 16–18). Data marked with a double asterisk are significantly different, based on a Student’s t test (0.001 ≤ P < 0.01).

Figure 2

Defensive properties of Arabidopsis plants in response to β-ocimene at different timings during plant development. Col-0 plants were exposed to the headspace volatiles released from TEC with (+) or without (−) 200 µg of β-ocimene at different times during their development, that is, during 7–13 dap, 16–22, 21–27, or 26–32 dap. A S. litura larva was released onto the plant at 32 dap and the net body weight of the larva gained during 4 d was determined (B). The experimental design is illustrated in (A). Data in (B) represent the mean and standard error (n = 18–20). Data marked with a double asterisk are significantly different, based on a Student’s t test (0.001 ≤ P < 0.01).

Figure 3

Transition of histone acetylation and transcriptional levels for ERFs. The Col-0 plants were exposed to the headspace volatiles released from TEC with (+) or without (−) β-ocimene during 7–13, 16–22, 21–27, or 26–32 dap (see Figure 2A). Relative acetylation levels of histone H4 (A) and relative transcript accumulation levels (B) of ERF8, ERF72, and ERF104 in leaves at 32 dap were determined. Data represent the mean and standard error (n = 3 (A) and n = 4–6 (B)). Data marked with an asterisk are significantly different, based on a Student’s t test (*0.01 ≤ P < 0.05).

Figure 4

Histone acetylation and transcriptional levels for ERFs immediately after exposure to β-ocimene. Col-0 plants were exposed to the headspace volatiles released from TEC with (+) or without (−) β-ocimene at different times during 7–13, 16–22, or 21–27 dap (see Figure 2A). Relative acetylation levels of histone H4 (A) and relative transcript accumulation levels (B) of ERF8, ERF72, and ERF104 in leaves at the respective endpoints were determined. Data represent the mean and standard error (n = 3 (A) and n = 4–6 (B)). Data marked with an asterisk(s) are significantly different, based on a Student’s t test (**0.001 ≤ P < 0.01; *0.01 ≤ P < 0.05). Otherwise, the means shown with a P-value are marginally different from those of the control.

Figure 5

Involvement of HATs in the β-ocimene response. A, Effect of HAT inhibitor (garcinol) treatment on relative transcript levels of ERF8 and ERF104 in leaves of WT Col-0 plants exposed to the headspace volatiles released from TEC with or without β-ocimene during 26–32 dap (see Figure 2A). B and C, Relative transcript levels (B) and relative acetylation levels of histones H3 and H4 (C) of ERFs in leaves of WT and T-DNA insertion mutant plants of the respective HATs, after exposure to the headspace volatiles released from TEC with (+) or without (−) β-ocimene. Data represent the mean and standard error (n = 3–5, 3–5, and 3–4 for (A), (B), and (C), respectively). For (A), means indicated by different small letters are significantly different, based on an ANOVA with post hoc Tukey’s HSD (P < 0.05). For (B) and (C), data marked with an asterisk(s) are significantly different between (+) and (−), based on a Student’s t test (**0.001 ≤ P < 0.01; *0.01 ≤ P < 0.05). Otherwise, the means shown with a P-value are marginally different from those of the control.

Figure 6

Defensive properties of HAT-deficient mutant plants in response to β-ocimene. WT Col-0 plants and T-DNA insertion mutant plants of the respective HATs were exposed to the headspace volatiles released from TEC without (−) or with (+) β-ocimene during 26–32 dap (see Figure 2A). A S. litura larva was released onto the plants at 32 dap, and the net body weight that the larva gained during 4 d was determined. Data represent values relative to those from WT plants with volatiles from TEC alone, with the mean ± standard error (n = 10–12). Data marked with an asterisk(s) are significantly different between (+) and (−), based on a Student’s t test (**0.001 ≤ P < 0.01; *0.01 ≤ P < 0.05).

Figure 7

Involvement of HDA6 in transcriptional regulation of ERF8 and ERF104 following defense induction. Relative transcript accumulation levels (A) and acetylation levels of histones H3 and H4 (B) of ERF8 and ERF104 in leaves of WT Col-0 and hda6 mutant plants exposed to the headspace volatiles released from TEC were determined. C, Positional profiles of histone H4 acetylation on ERF8 and ERF104 in leaves of WT and hda6 plants exposed to volatiles from TEC with (+) or without (−) β-ocimene, during 26–32 dap (see Figure 2A). D, A S. litura larva was released onto the plant immediately after the WT and hda6 plants were exposed to the headspace volatiles released from TEC ± β-ocimene, and the net body weight of the larva gained during 4 d was determined. Data represent the mean and standard error (n = 5, 3 and 23 for (A), (B) and (D), respectively). For (A) and (B), data marked with an asterisk(s) are significantly different, based on a Student’s t test (**0.001 ≤ P < 0.01; *0.01 ≤ P < 0.05). For (D), means indicated by different small letters are significantly different, based on an ANOVA with post-hoc Tukey’s HSD (P < 0.05).

Figure 8

Model of the histone acetylation/deacetylation on ERF8/ERF104, leading to defense responses in Arabidopsis plants in response to β-ocimene.