Measurements
These measurements correspond to the results of the biochemical characterization of plants overexpressing the PAP1 gene and plants exposed to volatile compounds. To confirm our hypothesis, after irradiating our plants with UV-B light for 2 hours and a half we followed the anthocyanins and carotenoids protocol to determine the levels of these photoprotective pigments. This way, we were able to verify if a higher accumulation of photoprotective pigments results in higher protection of the plants to UV-B light.
To start with the anthocyanins protocol, we collected the leaves two days after treatment and ground them in liquid nitrogen with a pestle and mortar until making green dust. Then to extract the pigments, we divided the leaf dust into different Eppendorf in which we added the MFV (methanol:formic:water) buffer. After a day of reaction at 4ºC, we centrifuged it to obtain the pellet and we separated the supernatant keeping it apart. After repeating the process another time, we prepared two Eppendorf for each sample, one with pH 1 solution and the other one with pH 4.5 solution, adding supernatant in each sample. Finally, after waiting for two hours, we measured absorbances at different wavelengths (512 and 700 nm) with the spectrophotometer to obtain our results using specific formulas.
On the other hand, for the carotenoids protocol, we followed the first steps of the previous protocol but we mixed the supernatant with acetone 80%. Afterwards, we measured the absorbances again at four different wavelengths (710, 663, 646 and 470 nm) to obtain our final results.
Once we obtained the samples’ absorbances we proceeded to analyze the results. Through different formulas based on a linear calibration curve, we transformed these absorbances in carotenoids and anthocyanins concentration in mg/L.
Grounding the leaves
Pigment extracts
Spectrophotometer
QUANTIFICATION OF ANTHOCYANINS
According to anthocyanin absorbance, the following formula was used to obtain the levels of anthocyanins:
?A = (A512-A700) pH 1 - (A512-A700) pH 4.5
Where ?A is the difference in absorbance between the anthocyanin extract diluted in pH 1.0 and pH 4.5 buffers; as from pH 4 onwards, these pigments do not show their color. In addition to this, the A700 nm was employed in the calculation of ?A to correct any background absorbance due to turbidity on the extracts.
Finally, to transform these absorbances into the concentration of anthocyanins in mg/L, y = 0,0114x - 0,008 calibration was used.
Calibration of Cyanidin-3glucoside for anthocyanin quantification.
The results obtained,represented in these tables, as mg equivalents of cyanidin-3glucoside per g FW, lead us to compare the levels of this pigment in WT and PAP1 plants and in wt plants grow in the presence or absence of A. alternata cultures after being irradiated with UV-B light to prove if our hypothesis was correct.
These results will be explained and discussed in the section of "Graphics and visual exam".
QUANTIFICATION OF CAROTENOIDS
According to carotenoid absorbance spectrum and considering the interference of chlorophyll absorbance at carotenoid absorbance wavelength (470 nm), the following formulas, described by Lichtenthaler, H. K., & Buschmann, C. (2001), were used to obtain the levels of carotenoids:
Chlorophyll a = 12,25 A663 – 2,79 A646
Chlorophyll b = 22,9 A646 – 4,68 A663
Chlorophyll a + b = 8,02 A663 – 20,20 A646
Total carotenoids =(1000 A470 - 1,63 Chlorophyll a-85,02 Chlorophyll b )/198
The A710 nm was used to control the turbidity on the extracts.
This way, we obtain just the levels of this photoprotective pigment, carotenoids, in WT and PAP1 plants and in wt plants grow in the presence or absence of A. alternata cultures.
The results presented in these tables will be explained and discussed in the section of "Graphics and visual exam".