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The exudate of
Chemical structures of diterpenes in the exudate of
The great ecological interest in plant terpenoids is reflected in several reviews of their ecological chemistry and role [
In particular, the objective was to assess how terpenoid levels vary naturally over a long period of time, and to evaluate the environmental factors responsible for their synthesis to determine and inquire more deeply into the possible role that these compounds are playing.
Amounts (mg/g dryw) of diterpenes (D1: diterpene 1; D2: diterpene 2; D3: diterpene 3) in leaves collected in autumn, winter, spring, and summer in the selected populations. The values are the means of the four populations studied over two years. SD: standard deviation.
Spring  Summer  Autumn  Winter  ANOVA  



3.50 ^{a}  3.28 ^{a}  3.33 ^{a}  8.03 ^{b}  
SD  0.73  2.04  1.04  0.77  

2.76 ^{a}  2.03 ^{a}  2.38 ^{a}  4.10 ^{b}  
SD  1.09  1.03  0.67  0.64  

6.51 ^{b}  3.25 ^{a}  3.56 ^{a}  8.79 ^{c}  
SD  2.36  0.98  1.51  3.11  

12.78 ^{a}  8.57 ^{a}  9.28 ^{a}  20.93 ^{b} 
^{a, b, c}: Same letter denote the absence of statistical differences (Tukey test).
There are very few studies that quantify the amount of diterpenes in Mediterranean shrubs. Most studies are focused on quantifying the emissions of aromatic terpenes such as monoterpenes [
Statistical analysis of the results (twoway ANOVA) to test the influence of temperature and water stress (independent variables) on the amount of diterpene in the leaves (dependent variable).






Corrected Model  51659.677 ^{a}  3  17219.892  3.756  0.025 
Intercept  632425.504  1  632425.504  137.955  0.000 
Temperature  31066.47  1  31066.47  6.777  0.016 
Stress  20447.496  1  20447.496  4.46  0.046 
Temperature * Stress  440.874  1  440.874  0.096  0.759 
^{a}: R Squared = 329 (Adjusted R Squared = 241); *: Interaction between the two variables.  






Corrected Model  47756.165 ^{a}  3  15918.722  7.45  0.001 
Intercept  523104.439  1  523104.439  244.799  0.000 
Temperature  33921.278  1  33921.278  15.874  0.001 
Stress  13645.168  1  13645.168  6.386  0.019 
Temperature * Stress  155.981  1  155.981  0.073  0.789 
^{a}: R Squared = 493 (Adjusted R Squared = 427); *: Interaction between the two variables.  






Corrected Model  47555.955 ^{a}  3  15851.985  9.31  0.000 
Intercept  383756.337  1  383756.337  225.394  0.000 
Temperature  45822.766  1  45822.766  26.913  0.000 
Stress  8.728  1  8.728  0.005  0.944 
Temperature * Stress  2.305  1  2.305  0.001  0.971 
^{a}: R Squared = 548 (Adjusted R Squared = 490); *: Interaction between the two variables. 
Percentage variation relative to the beginning of the trial in the amount of diterpenes in
In particular, under these conditions the amount of diterpene D1 increased by 115%, D2 by 99%, and D3 by 64%. The plants subjected to high temperatures and water stress presented reductions in formation of the three diterpenes but it was not significant. Low temperatures and water stress, however, again led to increased synthesis, but less than that with low temperatures and no water stress. For the low temperature trials there were no significant differences in diterpene content between the plants maintained with and without water stress, demonstrating that temperature is the determinant factor in the synthesis of these compounds.
These results are coherent with those obtained under natural conditions, confirming the increase of diterpene in winter, the season when the lowest temperatures are reached. This response of
Previous studies have demonstrated that phenol biosynthesis in
For this study, four populations of
Values for the four populations selected for sample collection of: T_{max}: mean of the seasonal maximum temperatures (°C); T_{min}: mean of the minimum seasonal temperatures (°C); P: total seasonal rainfall (mm). Location UTM coordinates.
Quintana  Hornachos  Jerez de los  Cabeza  

de la Serena  Caballeros  la Vaca  

30 S 262444  29 S 754337  29 S 685866  29 S 728663  
E 4289018  E 4273087  E 4238589  E 4219514  

25.5  24.2  24.3  25.1  
11.3  12.2  11.1  10.9  
113  139.8  126  155  

34.2  32.7  33.3  34.7  
18.1  16.3  17.4  18.3  
38  44  40  5  

16.8  17.8  15.6  15.2  
8.4  7.9  8.7  8.8  
156  219  300  309  

14  13.9  14.4  13.9  
5.5  6.5  5.2  4.9  
130  130  160  187 
Four trials were designed under controlled conditions (in a culture room) of high and low temperatures with and without water stress. All the trials maintained the same photoperiod (14 hours of light). Each trial was conducted on 9 individuals. The conditions of the different trials were the following:
Trial A: High temperatures (30 °C light; 15 °C dark), without water stress (plants watered every day);
Trial B: High temperatures, with water stress (plants moderately watered every 10 days);
Trial C: Low temperatures (13 °C light; 4 °C dark), without water stress;
Trial D: Low temperatures, with water stress.
The temperatures were selected as the means of the summer maxima (30 °C), summer minima (15 °C), winter maxima (13 °C), and winter minima (4 °C) where the distribution of
Approximately 0.5 g of leaves (2–3 leaves) was dipped several times into 2 mL of chloroform (5 replicates for each determination) [
Quantification of diterpenes in leaf was made by HPLC (Waters, 515 HPLC Pump, 717 plus Autosampler Injector, 996 Photodiode Array Detector). Aliquots of 25 µL were injected into a Spherisorb 5 µ C18 4.6 × 250 mm reverse phase analytical column. The mobile phase used was water/acetonitrile, with the following gradient: 0–5 min: 100% water (1 mL/min); 5–30 min: 70/30 wateracetonitrile (1 mL/min); 30–65 min: 45/55 wateracetonitrile (1 mL/min); 65–75 min: 100% acetonitrile (1 mL/min); 75–85 min: 100% water (1 mL/min). Once the chromatogram had been obtained (
HPLC chromatogram of diterpenes extracts captured at 260 nm. D1: 6acetoxy7oxo8labden15oic acid; D2: 7oxo8labden15oic acid; D3: oxocativic acid.
To obtain these equations, the different
Calibration equations:
D1 linear calibration equation: y = 1.1186x; r^{2} = 0.988;
D2 linear calibration equation: y = 0.9307x; r^{2} =0 .998;
D3 linear calibration equation: y = 1.3238x; r^{2} = 0.992.
All variables were tested for normality (ShapiroWillk test). Parametric tests were used to demonstrate the normality of variables. ANOVA test was used to test differences between seasons and
To conclude, we have shown in this study that the amount of flavonoids present in the leaves is markedly seasonal, and it is dependent on climate characteristics, principally the temperature, but in different way than flavonoids. Low temperatures increase the amount of diterpene in the leaves, while higher temperatures increase the amount of flavonoids as demonstrated in previous studies. These differences between the components of exudate suggest the involvement of diterpenes in different ecological functions.
This research was supported by: Consejería de Educación, Ciencia y Tecnología (Junta de Extremadura), project number: 3PRO5A084.