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.
Table 1.
Gene expression of G. biloba leaves after 4 h from S. littoralis herbivory.
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.
Table 2.
Comparative analysis of flavonoids, bilobalide and ginkolides between mechanically damaged (MD) and Spodoptera littoralis wounded (HW) Ginkgo biloba leaves after 4 h feeding.
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.
Table 3.
Analysis of VOCs in the headspace of treated G. biloba leaves.