Changes in cytokinins are sufficient to alter developmental patterns of defense metabolites in Nicotiana attenuata

Abstract

Plant defense metabolites are well known to be regulated developmentally. The optimal defense (OD) theory posits that a tssue's fitness values and probability of attack should determine defense metabolite allocations. Young leaves are expected to provide a larger fitness value to the plant, and therefore their defense allocations should be higher when compared with older leaves. The mechanisms that coordinate development with defense remain unknown and frequently confound tests of the OD theory predictions. Here we demonstrate that cytokinins (CKs) modulate ontogeny-dependent defenses in Nicotiana attenuata. We found that leaf CK levels highly correlate with inducible defense expressions with high levels in young and low levels in older leaves. We genetically manipulated the developmental patterns of two different CK classes by using senescence- and chemically inducible expression of CK biosynthesis genes. Genetically modifying the levels of different CKs in leaves was sufficient to alter ontogenic patterns of defense metabolites. We conclude that the developmental regulation of growth hormones that include CKs plays central roles in connecting development with defense and therefore in establishing optimal patterns of defense allocation in plants.

Keywords: Manduca sexta; Nicotiana attenuata; cytokinins; herbivores; immunosenescence; inducible defense; optimal defense; phytohormones; plant development.

Fig. 1. Herbivory-induced defense metabolites (HIDs) and cytokinins (CKs) follow the same within-plant distributions in Nicotiana attenuata.

(A) Experimental design and distribution of HID and CKs within a plant. (B) Scheme of biosynthetic pathway of major phenylamides. (C) Caffeoylputrescine; not detectable (n.d) in control leaves. (D) Relative transcript abundance of transcription factor NaMYB8 and (E) NaAT1 as well as (F) CKs (cis-zeatin, cZ; cis-zeatin riboside, cZR; dihydrozeatin, DHZ; dihydrozeatin riboside, DHZR; isopentenyladenine, IP; isopentenyladenosine, IPR; trans-zeatin, tZ; trans- zeatin riboside, tZR; other CKs in table S1), in different leaf-classes of flowering plants: rosette leaves R-1 (youngest) to R-4 (oldest) and stem leaves S+1 (oldest) to S+4 (youngest). Plants were sprayed for two days with 1 mM methyl jasmonate (2 d MJ; dotted bars) or water as control (open bars). Data were analyzed by two-way ANOVAs (D, E) or one-way ANOVAs (C), p-values indicate influence of the single factors leaf and MJ-treatment or the interaction of both (Leaf * MJ-treat.). Statistics for CKs can be found in table S2. Error bars depict standard errors (N ≥ 5). FM, fresh mass.

Fig. 2. Herbivory-induced defense metabolites (HIDs) and cytokinins (CKs) follow similar developmental patterns in Nicotiana attenuata.

(A) Experimental design and the distribution of HIDs and CKs during plant development. (B) Typical damage after 3 d Manduca sexta feeding and control leaf. (C) Caffeoylputrescine, (D) relative transcript abundance of transcription factor NaMYB8 and (E) NaAT1 as well as (F) CKs (cis-zeatin, cZ; cis-zeatin riboside, cZR; dihydrozeatin, DHZ; dihydrozeatin riboside, DHZR; isopentenyladenine, IP; isopentenyladenosine, IPR; trans-zeatin, tZ; trans-zeatin riboside, tZR; other CKs in table S5) in the same leaf position (young rosette leaf) in two growth stages: vegetative rosette plants and reproductive flowering plants. Open bars: control levels, diagonally striped bars: levels after 3 d M. sexta feeding. Two-way ANOVAs, p-values indicate influence of the single factors growth-stage (GS) and M. sexta (M.s.) feeding or the interaction of both (GS * M.s. feeding). Statistics for CKs can be found in table S6. Different letters indicate significant differences (if interaction was significant: Tukey HSD post hoc test: p<0.05). Error bars depict standard errors (N ≥ 9). FM, fresh mass.

Fig. 3. Correlations of cytokinin levels in untreated leaves with the accumulations of different defense compounds after MJ-induction in Nicotiana attenuata.

Average levels of caffeoylputrescine, dicaffeoylspermidine, NaTPI transcript levels and nicotine in different leaf types of a flowering plant after induction with MJ plotted against levels of CKs (isopentenyladenine, IP; isopentenyladenosine, IPR; trans-zeatin, tZ; trans-zeatin riboside, tZR) in uninduced leaves at the same leaf-position. Error bars depict standard errors (N ≥ 5). FM, fresh mass.

Fig. 4. Manipulation of the within-plant cytokinin gradient alters the distribution of herbivory-inducible defenses in Nicotiana attenuata.

(A) Experimental design. (B) CKs: cis-zeatin, cZ; cis-zeatin riboside, cZR; dihydrozeatin, DHZ; dihydrozeatin riboside, DHZR; isopentenyladenine, IP; isopentenyladenosine, IPR; trans-zeatin, tZ; trans-zeatin riboside, tZR; other CKs in table S7) and (C) relative transcript abundance of transcription factor NaMYB8 and (D) NaAT1 and (E) caffeoylputrescine in different leaf-classes (Rosette leaves 4-6, R-4-6,rosette leaf 3, 2 and 1 with R-1 being the youngest and R-6 being the oldest, first 3 stem leaves 1-3 (S+1-3) and stem leaves 4-6 (S+4-6)) of flowering plants transformed with a construct for dexamethasone-inducible expression of the CK biosynthesis enzyme isopentenyltransferase (i-ovIPT). R-2 was treated with 5 µM dexamethasone and 1% DMSO in lanolin paste (DEX; red color; ↑CK) to increase levels of tZ-type CKs in the leaves or with 1% DMSO in lanolin as control (Mock, white color). All other leaves were mock-treated. Grey bars indicate levels from plants in which one leaf was DEX-treated. Plants were sprayed for two days with 1 mM methyl jasmonate (MJ). p-value above brackets over R-2 leaves represent results of a t-test between DEX- and mock-treated R-2 leaves. Asterisks in different sections of CK-bars represent statistically significant differences (p<0.05) from t-tests between single CKs. Error bars depict standard errors (N ≥ 4); FM, fresh mass.

Fig. 5. Restoring cytokinin levels to an earlier developmental stage increases defense gene expression and recovers inducibility of defenses in flowering Nicotiana attenuata plants.

Flowering wildtype (WT) and SAG-IPT4 plants. (A) CKs (cis-zeatin, cZ; cis-zeatin riboside, cZR; dihydrozeatin, DHZ; dihydrozeatin riboside, DHZR; isopentenyladenine, IP; isopentenyladenosine, IPR; trans-zeatin, tZ; trans-zeatin riboside, tZR; other CKs in table S10), (B) relative transcript abundance of transcription factor NaMYB8 and (C) NaAT1 and (D) caffeoylputrescine. Levels were measured in the youngest rosette leaf after three days of Manduca sexta feeding (3 d M. sexta feeding, diagonal striped bars) and in control leaves of unattacked plants (control; open bars). Data were analyzed by two-way ANOVAs (C) or generalized least squares models (B, D), p-values indicate influence of the single factors genotype (line) and M. sexta (M.s.) feeding or the interaction of both (Line * M.s. feeding). Different letters indicate significant differences (if interaction was significant: pairwise Wilcoxon rank-sum test with Bonferroni correction (B, D): p<0.05). Asterisks in different sections of active CK-bars indicate significant differences (p<0.05) in t-tests with single CKs between control and induced levels of different genotypes respectively (t-tests; * p < 0.05, ** p < 0.01). Results of two-way ANOVAs of CKs can be found in table S11. Results for line SAG-IPT4-2 can be found in tables S10 – S15. Error bars show standard errors (N ≥ 5). FM, fresh mass.

Fig 6. Cytokinins (CKs) influence the developmentally dependent distribution of defense metabolites in Nicotiana attenuata.

Black arrows: findings of this paper; grey arrows: previous publications as indicated next to the arrow. Leaf ontogeny/its developmental state change levels of CKs and vice versa. CK levels change levels of herbivory-inducible defenses. How exactly CKs influence herbivory-induced defenses remains to be discovered. We found evidence for transcriptional and post-transcriptional regulation. The main conclusion of McKey’s Optimal Defense Theory is highlighted by the light green box: investment in defense metabolism in a tissue depends on its value and probability of attack. We hypothesize that leaf value and probability of attack are also influenced by growth hormones, such as CKs.