Ginkgo biloba responds to herbivory by activating early signaling and direct defenses

Abstract

Background: Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses.

Methodology/principal findings: Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca(2+)](cyt)) and H(2)O(2) production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca(2+)](cyt) and H(2)O(2). Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides.

Conclusions/significance: By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this "living fossil" plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.

Figure 1. G. biloba is characterized by different leaf types, depending on the age and shape of the leaf.

A, bilobed; B, multi-dissected and; C, fan-shaped. Vm values are reported along with standard errors (in brackets) as mV (n≈50). Herbivore wounding is shown in leaf segments and Vm values are indicated below and aside the wounding zone. The leaf section of C shows Vm values of the different mesophyll and epidermal cells of a fan-shaped leaf. D, Spodoptera littoralis feeding on G. biloba leaves.

Figure 2. Calcium variations in G. biloba upon mechanical damage and herbivore wounding.

A. Mechanically wounded G. biloba leaves, values (n = 5) are expressed as µM Ca²⁺ calculated from a calibration curve. The same letter indicates not significant (P>0.05) variation. B. Herbivore wounded G. biloba leaves, values (n = 5) are expressed as µM Ca²⁺. Different letters indicate significant (P<0.05) differences, the asterisks indicate significant (P<0.05) differences with respect to mechanical damage. In both panels, calcium orange indicates the absence of pharmacological inhibitors.

Figure 3. H₂O₂ variations in G. biloba upon mechanical damage and herbivore wounding.

A. Mechanically-wounded G. biloba leaves, values (n = 5) are expressed as µM H₂O₂ calculated from a calibration curve. The same letter indicates not significant (P>0.05) variation. B. Herbivore-wounded G. biloba leaves, values (n = 5) are expressed as µM H₂O₂. Different letters indicate significant (P<0.05) differences, the asterisk indicate significant (P<0.05) differences with respect to mechanical damage. In both panels, amplex indicates the absence of pharmacological agents.

Figure 4. Subcellular localization of [Ca²⁺]_cyt and H₂O₂ in G. biloba leaves upon herbivory.

A. False color images from confocal laser scanning microscopy shows that upon herbivory [Ca²⁺]_cyt was found mainly in the cytosol, indicated by the calcium orange dye as green patches not associated with any specific organelle. Metric bar = 10 µm. B. H₂O₂ localization by Amplex Red shows a clear associations with microbodies (probably peroxisomes) and/or mitochondria but not with chloroplasts. Metric bar = 20 µm. In both panels, single arrows indicate the dye, double arrows indicate chloroplasts.

Figure 5. Time-course quantitative gene expression of some ROS scavenging genes in G. biloba upon herbivory.

Gene expression of superoxide dismutase (SOD) and catalase (CAT) was up-regulated by herbivory at all times. Upon herbivory, peroxidase (POX) was significantly down-regulated after 4 h, whereas ascorbate peroxidase (APX) was down-regualted after 30 min. The dotted lines represent control values (mechanical damage), different letters indicate significant (P<0.05) differences, asterisk indicates significant (P<0.05) differences with respect to control.

Figure 6. Structure formulae of the main representative G. biloba compounds.

Figure 7. Time-course quantitative gene expression of some G. biloba genes involved in phenylpropanoid and terpenoid metabolism upon herbivory.

Phenylalanine ammonia lyase (PAL) and anthocyanidin reductase (ANR) were significantly up regulated by herbivory only after 4 h, whereas flavonol synthase (FLS) was down-regulated at 30 min and 4 h. Chalcone synthase (CHS) showed a constant up-regulation, whereas flavanone 3-hydroxylase (F3H) showed an increased up regulation after 4 h. Farnesyl diphosphate synthase (FPPS) was significantly upregulated only after 30 min whereas geranylgeranyl diphosphate synthase (GGPP) showed no regulation at all times. Levopimaradiene synthase (PPS) was significantly down-regulated at all times. The dotted lines represent control values (mechanical damage), different letters indicate significant (P<0.05) differences, asterisks indicate significant (P<0.05) differences with respect to control.