Dynamika akumulace nestrukturních sacharidů a aktivity enzymu Rubisco při zvýšené koncentraci oxidu uhličitého a manipulaci sinku u buku lesního
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Vysoké učení technické v Brně. Fakulta chemická
Abstract
Tato diplomová práce se zabývá dynamikou akumulace nestrukturních sacharidů a aktivity enzymu Rubisco při zvýšené koncentraci CO2 u buku lesního (Fagus sylvatica L.). Tři roky staré sazenice buku lesního byly kultivovány čtyři měsíce v minisféře s běžnou (385 mol•mol-1, varianta A) a se zvýšenou koncentrací CO2 (700 mol•mol-1, varianta E). V každé variantě byla polovina rostlin hnojena dusíkem (varianta N+) a druhá polovina byla kontrolní (varianta N-). Rostliny použité pro experiment byly nejprve adaptovány na tmu po dobu 12 hodin. Následně byly testované listy odstřiženy, řapík včetně krátkého segmentu větvičky (asi 1 cm) pak vložen do 0,7 M roztoku sacharosy (varianta S) resp. vody (varianta V) a vystaveny záření 200 mol•m-2•s-1 po dobu 0, 30, 60, 120, 180 min. Poté byla stanovena listová plocha a čerstvá hmotnost listové čepele, vzorky byly fixovány v kapalném dusíku a dále až do samotné analýzy uchovávány v hlubokomrazícím boxu při teplotě –70 °C. Obsah Rubisco byl stanoven metodou SDS-PAGE, aktivita Rubisco spektrofotometricky a obsah nestrukturních sacharidů antronovou metodou a metodou HPLC. U varianty N- byl zjištěn statisticky významně nižší obsah Rubisco, než u varianty N+. Rovněž obsah Rubisco u varianty E byl významně nižší než u varianty A, což svědčí o aklimační depresi obsahu Rubisco. Aktivita Rubisco je s časem u varianty E mírně stimulována, ale rozdíly mezi variantami A a E nejsou statisticky významné. Vliv přídavku sacharosy na aktivitu Rubisco se nepodařilo prokázat. Antronovou metodou byly zjištěny statisticky významné rozdíly v akumulaci nestrukturních sacharidů mezi variantami S a V, ale nikoliv mezi variantami A a E. Metodou HPLC byl zjištěn statisticky významný nárůst obsahu sacharosy s časem u varianty AS, ale nikoliv u varianty ES.
This diploma thesis deals with dynamic of accumulation of non-structural carbohydrates and activity of Rubisco enzyme at elevated concentration of CO2 on beech (Fagus sylvatica L.). Three years old seedlings of beech were cultivated in minisphere with ambient (385 µmol•mol-1, variant A), and with elevated concentration CO2 (700 µmol•mol-1, variant E) for four months. In every variant the first half of plants was fertilized by nitrogen (variant N+) and the second half was control (variant N-). Plants used for experiment were at first adapted for darkness for 12 hours. Subsequently tested leaves were cut off, leafstalk including short segment of branch (approximately 1 cm) was inserted into 0.7 M solution of sucrose (variant S) or water (variant V) and exposed to radiation 200 mol•m-2•s-1 for 0, 30, 60, 120, 180 minutes. Then leaf area and fresh mass of leaf blade were established, samples were fixed in liquid nitrogen and stored in deep freezer to analysis in –70 °C. Rubisco content was determined by SDS-PAGE method, Rubisco activity spectrofotometrically and content of non-structural carbohydrates by anthrone method and HPLC method. Rubisco content was significantly lower in the N- variant than in N+ variant. Rubisco content was also significantly lower in E than in A variant, which is an evidence of down-regulation. Rubisco activity is moderately stimulated at E variant with time, but differences between variant A and E are not statistically significant. Influence of sucrose feeding to Rubisco activity was not proved. Significant differences were detected by anthrone method in non-structural carbohydrates content between variants S and V, but not between variants A and E. Statistically significant increase of sucrose content with time was detected by HPLC method at variant AS, but not at variant ES.
This diploma thesis deals with dynamic of accumulation of non-structural carbohydrates and activity of Rubisco enzyme at elevated concentration of CO2 on beech (Fagus sylvatica L.). Three years old seedlings of beech were cultivated in minisphere with ambient (385 µmol•mol-1, variant A), and with elevated concentration CO2 (700 µmol•mol-1, variant E) for four months. In every variant the first half of plants was fertilized by nitrogen (variant N+) and the second half was control (variant N-). Plants used for experiment were at first adapted for darkness for 12 hours. Subsequently tested leaves were cut off, leafstalk including short segment of branch (approximately 1 cm) was inserted into 0.7 M solution of sucrose (variant S) or water (variant V) and exposed to radiation 200 mol•m-2•s-1 for 0, 30, 60, 120, 180 minutes. Then leaf area and fresh mass of leaf blade were established, samples were fixed in liquid nitrogen and stored in deep freezer to analysis in –70 °C. Rubisco content was determined by SDS-PAGE method, Rubisco activity spectrofotometrically and content of non-structural carbohydrates by anthrone method and HPLC method. Rubisco content was significantly lower in the N- variant than in N+ variant. Rubisco content was also significantly lower in E than in A variant, which is an evidence of down-regulation. Rubisco activity is moderately stimulated at E variant with time, but differences between variant A and E are not statistically significant. Influence of sucrose feeding to Rubisco activity was not proved. Significant differences were detected by anthrone method in non-structural carbohydrates content between variants S and V, but not between variants A and E. Statistically significant increase of sucrose content with time was detected by HPLC method at variant AS, but not at variant ES.
Description
Citation
UHROVÁ, L. Dynamika akumulace nestrukturních sacharidů a aktivity enzymu Rubisco při zvýšené koncentraci oxidu uhličitého a manipulaci sinku u buku lesního [online]. Brno: Vysoké učení technické v Brně. Fakulta chemická. 2012.
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Document version
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Language of document
cs
Study field
Potravinářská chemie a biotechnologie
Comittee
prof. Ing. Peter Šimko, DrSc. (předseda)
doc. Ing. Jiřina Omelková, CSc. (místopředseda)
prof. RNDr. Ivana Márová, CSc. (člen)
doc. RNDr. Alena Španová, CSc. (člen)
doc. Ing. Bohuslav Rittich, CSc. (člen)
Date of acceptance
2012-06-05
Defence
1. Diplomantka seznámila členy komise s náplní a cílem diplomové práce.
2. Byly přečteny posudky na diplomovou práci.
3. Diplomantka akceptovala všechny připomínky oponenta a na všechny otázky odpověděla v plné šíři.
Diskuse:
doc. Márová: Jak souvisí vybrané cukry a enzym rubisco ?
Result of defence
práce byla úspěšně obhájena
Document licence
Standardní licenční smlouva - přístup k plnému textu bez omezení