R2R3-NaMYB8 regulates the accumulation of phenylpropanoid-polyamine conjugates, which are essential for local and systemic defense against insect herbivores in Nicotiana attenuata

Abstract

Although phenylpropanoid-polyamine conjugates (PPCs) occur ubiquitously in plants, their biological roles remain largely unexplored. The two major PPCs of Nicotiana attenuata plants, caffeoylputrescine (CP) and dicaffeoylspermidine, increase dramatically in local and systemic tissues after herbivore attack and simulations thereof. We identified NaMYB8, a homolog of NtMYBJS1, which in BY-2 cells regulates PPC biosynthesis, and silenced its expression by RNA interference in N. attenuata (ir-MYB8), to understand the ecological role(s) of PPCs. The regulatory role of NaMYB8 in PPC biosynthesis was validated by a microarray analysis, which revealed that transcripts of several key biosynthetic genes in shikimate and polyamine metabolism accumulated in a NaMYB8-dependent manner. Wild-type N. attenuata plants typically contain high levels of PPCs in their reproductive tissues; however, NaMYB8-silenced plants that completely lacked CP and dicaffeoylspermidine showed no changes in reproductive parameters of the plants. In contrast, a defensive role for PPCs was clear; both specialist (Manduca sexta) and generalist (Spodoptera littoralis) caterpillars feeding on systemically preinduced young stem leaves performed significantly better on ir-MYB8 plants lacking PPCs compared with wild-type plants expressing high levels of PPCs. Moreover, the growth of M. sexta caterpillars was significantly reduced when neonates were fed ir-MYB8 leaves sprayed with synthetic CP, corroborating the role of PPCs as direct plant defense. The spatiotemporal accumulation and function of PPCs in N. attenuata are consistent with the predictions of the optimal defense theory: plants preferentially protect their most fitness-enhancing and vulnerable parts, young tissues and reproductive organs, to maximize their fitness.

Figure 1.

NaMYB8 responds differentially to M. sexta OS elicitation in N. attenuata. A, Technical replicate means ± se of NaMYB8 transcript relative abundances quantified with RT-qPCR from pooled samples using five independent control, W+W-elicited, and W+OS-elicited plants. The inset shows a detail of rapid transient accumulation of transcripts between 15 and 120 min after elicitation. B, Mean ± se levels of NaMYB8 transcripts in unelicited and 1-h W+OS-elicited local leaves of two independent homozygous ir-MYB8 and wild-type (WT) plants (n = 3). Asterisks represent significantly different transcript abundances between genotypes within the same treatment group at P < 0.001. nd, Not detected.

Figure 2.

NaMYB8 regulates the accumulation of CP and DCS in M. sexta-attacked leaves. M. sexta caterpillars were allowed to feed on N. attenuata wild-type (WT) and NaMYB8 transgenic plants for 4 d before harvesting samples for analysis. A, HPLC chromatograms obtained from wild-type and ir-MYB-810 M. sexta-fed rosette leaf methanolic extracts detected at 254-nm wavelength. B to G, Mean ± se concentrations of CP (B), DCS (C), CGA (D), rutin (E), nicotine (F), and DTGs (G) in wild-type, ir-MYB8-818, and ir-MYB8-810 leaves directly attacked by herbivores quantified by HPLC. Asterisks represent significant differences among the genotypes within the treatment group at P < 0.001 (n = 5). FM, Fresh mass; nd, not detected.

Figure 3.

Young systemically induced N. attenuata leaves and reproductive tissues accumulate high levels of CP upon OS elicitation of rosette leaves. Wild-type (WT) plants were germinated in sand and supplemented with nutrients dissolved in water to the roots. At each developmental stage, a single fully expanded rosette leaf at (+1) position was OS elicited, and 3 d later the samples from representative plant parts were collected and analyzed by HPLC. The dotted arrow shows the position of the locally W+OS-elicited rosette leaf at different stages of development. NaMYB8 transcripts, shown in the gray inset, were analyzed from sample tissues used for the determination of CP at the elongated stage. R, Root; SB, stem basal; SU, stem upper; SeL, senescent leaf; OL, old leaf; LL, locally W+OS-induced rosette leaf; SL, systemic rosette leaf; 1SL, first stem leaf; YL, young stem leaf; B, flower buds; EB, elongating flower buds; F, open flowers; GC, green capsules with seeds; RC, ripe capsules with seeds; FM, fresh mass; nd, not detected.

Figure 4.

NaMYB8-silenced plants show normal growth and reproductive fitness. A and B, CP (A) and DCS (B) contents in reproductive tissues of wild-type (WT) and ir-MYB8-810 transgenic lines determined by HPLC. B, Flower buds; EB, elongating flower buds; F, open flowers; GC, green capsules with seeds; FM, fresh mass; nd, not detected. C, Mean ± se stalk lengths measured throughout plant development in the glasshouse, starting from day 41 after germination of plants. Asterisks represent significantly different growth parameters between wild-type plants and two ir-MYB8 homozygous lines at specific time points at P < 0.01 (**) and P < 0.001(***; n = 12). D, Mean ± se lifetime seed capsules produced by plants determined 98 d after germination. No statistically significant differences were observed. E, Schematic representation of the seed capsule position on the uppermost lateral branch used for seed mass determinations. T0, Seed capsule located nearest to the branching point on the top most lateral branch; T1, most distal seed capsule on the same branch. F, Mean ± se seed mass in representative seed capsules at T0 and T1 positions measured in 98-d-old plants. No statistically significant differences were observed.

Figure 5.

Silencing NaMYB8 in N. attenuata makes plants vulnerable to insect herbivores. A, Mean ± se CP and DCS accumulation in locally W+OS-induced rosette leaves and systemically induced young stem leaves in wild-type N. attenuata analyzed 3 d after induction (n = 4); control plants remained unelicited. Neither CP nor DCS was detected in ir-MYB8 plants. B, A pattern wheel-wounded rosette leaf of a wild-type plant was treated either with the OS-specific elicitor C18:3-Glu dissolved in 0.02% Tween or with 0.02% Tween (mock treatment), and both local and systemic accumulation of CP and DCS were analyzed as above. C, Mean ± se mass gained by N. attenuata generalist herbivore (S. littoralis) and specialist herbivore (M. sexta) caterpillars when fed on systemically preinduced young stem leaves of ir-MYB8-810 and wild-type (WT) plants, whose rosette leaves were elicited with C18:3-Glu (FAC). A single rosette leaf was elicited every 4th d to enhance the CP accumulation in the young stem leaves. Asterisks represent significantly different growth responses of herbivores that fed on wild-type and homozygous ir-MYB8-810 plants at specific time points at P < 0.01 (**) and P < 0.001 (***; n = 16). FM, Fresh mass.

Figure 6.

CP functions as an indispensable part of direct defense against M. sexta in N. attenuata. A, Mean ± se concentrations of CP sprayed on ir-MYB8-810 leaves over a period of 48 h. B, Mean ± se M. sexta caterpillar mass gain 4 d after feeding on CP-sprayed or water-sprayed ir-MYB8-810 leaves. The asterisk represents significantly different M. sexta caterpillar growth responses between the treatments at P < 0.05 (n = 16). FM, Fresh mass.

Figure 7.

NaMYB8-regulated metabolic pathways in N. attenuata plants. The NaMYB8 transcription factor transcriptionally controls the accumulation of CP and DCS metabolites in N. attenuata plants. NaMYB8-dependent genes determined by microarray analysis are enclosed in rectangles. ADC, Arg decarboxylase; C3H, cinnamic acid-3-hydroxylase; C4H, cinnamic acid-4-hydroxylase; CHS, chalcone synthase; 4CL, 4-coumaroyl-CoA:ligase; COMT, caffeic acid O-methyltransferase; HQT, hydroxycinnamoyl-CoA quinate transferase; ODC, Orn decarboxylase; PAL, Phe ammonia lyase; PMT, putrescine methyltransferase; SPS, spermidine synthase.