Thesis (M.Sc. in Biochemistry) -- Mahidol University, 1970.

No description available.

No description available.

No description available.

The lower oxygen limit (LOL) in plants marks the oxygen (O2) level where the metabolism shifts from being predominantly aerobic to anaerobic; recent work has shown that respiratory-based indicators of this metabolic shift are well-correlated with changes in chlorophyll a fluorescence signals. The physiological and biochemical changes at the root of this relationship have not been well-described in the literature. The processes involved are spatially separated: chlorophyll fluorescence is associated with the lightdependent reactions and emanates from the chloroplasts whereas aerobic respiration and fermentation occurs in the mitochondria and cytosol, respectively. Evidences outlined in this thesis are used to suggest the mechanistic link between these three regions of the cell is a fluid exchange of cellular reductant. When mitochondrial respiration is inhibited as a result of inadequate O2, used as a terminal electron acceptor, glycolytic reductant in the form of NADH accumulates in the cytosol. Reductant imbalances between the cytosol and organelles can be adjusted indirectly using translocators. Excess chloroplastic reductant is used to reduce the plastoquinone (PQ) pool via NADPH-dehydrogenase, a component of the chlororespiratory pathway, effectively decreasing the photochemical quenching (qP) capacity thereby inducing a switch from minimum fluorescence (Fo) to a higher relative fluorescence (F) value where qP < 1. Subjecting dark-adapted photosystems to low-intensity light increased Fo to a slightly higher F value due to a lightinduced reduction of the oxidized PQ pool when the O2 was above the LOL, but decreased F as a result of a PSI-driven oxidation of the already over-reduced PQ pool when the O2 was below the LOL. Low O2 was also shown to increase violaxanthin deepoxidation and non-photochemical quenching (qN), likely a reflection of the overreduced state of the photosystems and associated pH decrease. Dynamic controlled atmosphere (DCA) is a fluorescence-based controlled atmosphere (CA) system that sets the optimum atmosphere for fruits and vegetables based on a product’s fluorescence response. Experiments in this thesis on the relationship between O2, temperature, light, metabolism, pigmentation and chlorophyll fluorescence were used to interpret the physiology behind fluorescence changes, suggest improved DCA techniques and outline potentially profitable avenues for future research.

Der Abbau des Chlorophyll (Chl) ist ein Prozess, der typischerweise während der Blattseneszenz und der Reifung von Früchten und Samen stattfindet. Eine Störung dieses koordinierten Prozesses unter Frostbedingungen verzögert den Chl-Abbau und ist ein grosses Hindernis bei der Herstellung von hochwertigem Rapsöl. Der Abbau von Chl zu farblosen Kataboliten erfolgt in einer Serie von enzymatischen Schritten und wird durch die Chlorophyllase begonnen (Chlase). Es wurde vorgeschlagen, dass ein wasserlösliches Chl Protein (WSCP) den Transport des Chl von der Thylakoidmembran zum Wirkort der Chlase übernimmt. Weiterhin wurde angenommen, dass die Steigerungen der Genexpressionen dieser frühen Schritte den Prozess des Chl-Abbaus beschleunigen. In der vorliegenden Arbeit werden die Auswirkungen der Überexpression der Chlase aus Citrus clementii (CcCHLASE) und von WSCP aus Blumenkohl (Cau-WSCP) in transgenen Tabakpflanzen analysiert. Dazu wurde die cDNA Sequenz der CcCHLASE in E. coli exprimiert und mittels in vitro Experimenten die Hydrolysierung von Chl durch die Chlase bestätigt. Anschließend wurden CcCHLASE exprimierende Tabakmutanten generiert und drei T1-Linien wurden unter verschiedenen Stress- und Seneszenzbedingungen untersucht. Die Chlase überexprimierenden Linien zeigten unter allen getesteten Bedingungen einen im Vergleich zum Wildtyp erhöhten Chlide a Gehalt. Trotzdem unterschied sich die Menge an Endkataboliten in diesen Mutanten nicht vom Wildtyp. Andererseits zeigten WSCP überexprimierende Linien zwar keine erhöhten Chlide a Gehalte jedoch erhöhte Protochlorophyllid-(Pchlide)-Level. Das deutet auf eine Rolle des WSCP als Speichermolekül für Chlorophyllvorstufen hin. Die photoprotektive Funktion des WSCP wurde zusätzlich in WSCP überexprimierenden Linien bestätigt. Diese zeigen im Vergleich zu Wildtyp-Tabakpflanzen auch bei hohen Lichtintensitäten von 700 – 900 µmol Photonen m-2 s-1 verringerte Gehalte an Zeaxanthin und reduzierte Peroxidaseaktivitäten. / Chlorophyll (Chl) catabolism is characteristically seen during leaf senescence, fruit ripening and seed maturation. Disruption of this coordinated process under frost conditions delays Chl breakdown and is a great concern in rapeseed oil production. The present work addresses this problem by studying the effect of enhanced Chl catabolism in genetically modified tobacco plants. Chl is catabolised to colourless catabolites through a series of enzymatic reactions initiated by Chlorophyllase (Chlase). A water soluble chlorophyll protein (WSCP) has been proposed to transport Chl from thylakoid membranes to the site of action of Chlase. It was assumed that enhancing the gene expression of these early events in Chl catabolism would increase the Chl breakdown process. The present work analysed the overexpression of Chlase from Citrus clementii (CcCHLASE) and WSCP gene from cauliflower (Cau-WSCP) in modified tobacco plants. Initially, the cDNA sequence of CcCHLASE was expressed in E. coli and in vitro tests confirmed the hydrolytic activity of Chlase on Chl. Subsequently, tobacco plants overexpressing CcCHLASE were generated and three T1 lines were analysed at various stress and senescence conditions. The in vivo production of Chlorophyllide (Chlide) indicated the extent of increased Chl breakdown. The Chlase overexpressor lines showed higher Chlide a steady state levels under all tested conditions in comparison to the WT tobacco plants. However, the end catabolites did not show much difference from WT plants. On the other hand, WSCP overexpressor lines did not show any increase in Chlide a levels, but demonstrated an increased protochlorophyllide (Pchlide) levels. This suggested the role of WSCP as a storage molecule of Chl precursors. Additionally, photoprotective function of WSCP was confirmed in WSCP overexpressors, by lower zeaxanthin levels and peroxidase activity even at high light intensities of 700 – 900 µmol photons m-2 s-1 in comparison to the WT tobacco plants.

<p>In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy.</p><p>In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function.</p><p>I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained.</p><p>The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light.</p><p>Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role.</p><p>The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants.</p><p>A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.</p>

In photosynthesis, sunlight is converted into chemical energy that is stored mainly as carbohydrates and supplies basically all life on Earth with energy. In order to efficiently absorb the light energy, plants have developed the outer light harvesting antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a and b as well as different carotenoids. In addition to the light harvesting function, the antenna has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is crucial to avoid oxidative damage under conditions of high excitation pressure. Another regulatory function in the antenna is the state transitions in which the distribution of the trimeric LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are conserved in all higher plants and based on evolutionary arguments this has led to the suggestion that each protein has a specific function. I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained, in which the target proteins were reduced, in some cases beyond detection level, in other cases small amounts remained. The results show that CP29 has a unique function as organising the antenna. CP26 can form trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain growth rates in very low light. Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins clearly shows that there is no direct involvement of either CP29 or CP26 in this process. Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE. A model for feedback de-excitation is presented in which PsbS plays a major role. The positions of the minor antenna proteins in the PS II supercomplex were established by comparisons of transmission electron micrographs of supercomplexes from the wild type and antisense plants. A fitness experiment was conducted where the antisense plants were grown in the field and seed production was used to estimate the fitness of the different genotypes. Based on the results from this experiment it is concluded that each Lhcb protein is important, because all antisense lines show reduced fitness in the field.

Title from second page of PDF document. Includes bibliographical references.

Τα φύλλα κάποιων φυτών γίνονται παροδικά κόκκινα, λόγω της συσσώρευσης ανθοκυανινών. Εξετάστηκαν δύο από τις πολλές υποθέσεις που έχουν διατυπωθεί για το ρόλο των χρωστικών αυτών στα φύλλα: ο φωτοπροστατευτικός τους ρόλος και η εμπλοκή τους στις σχέσεις φυτών και φυτοφάγων εντόμων. Χρησιμοποιήθηκαν δύο είδη φυτών (Quercus coccifera και Cistus creticus) τα οποία παρουσιάζουν ενδοειδική ποικιλομορφία όσον αφορά στη συσσώρευση ανθοκυανινών στα φύλλα τους. Στο Q. coccifera, τα νεαρά φύλλα κάποιων ατόμων είναι κόκκινα και κάποιων άλλων πράσινα, ενώ κατά την ενηλικίωση τους γίνονται όλα πράσινα. Τα ώριμα φύλλα του C. creticus το καλοκαίρι είναι πράσινα (“πράσινη” περίοδος) αλλά συσσωρεύονται παροδικά ανθοκυανίνες στα ώριμα φύλλα κάποιων θάμνων κατά τη περίοδο του χειμώνα (“κόκκινη” περίοδος), ενώ γειτονικά άτομα παραμένουν πράσινα. Συγκρίθηκαν παράμετροι του in vivo φθορισμού της χλωροφύλλης, τα επίπεδα των συστατικών του κύκλου των ξανθοφυλλών και των ολικών φαινολικών, τα φάσματα ανακλαστικότητας και η ένταση της φυτοφαγίας, σε κόκκινα και πράσινα φύλλα από αντίστοιχους φαινοτύπους που αναπτυσσόντουσαν στο ίδιο ενδιαίτημα. Δεν διαπιστώθηκε κάποιο συγκριτικό πλεονέκτημα (ή μειονέκτημα) των ανθοκυανικών φύλλων όσον αφορά στη φωτοσυνθετική τους λειτουργία ή στην ανθεκτικότητά τους έναντι της φωτοαναστολής, στις συνθήκες που αναπτύσσονται. Στα διαφορετικού χρώματος, νεαρά φύλλα του Q. coccifera, είναι σαν να γίνεται ένας “συμβιβασμός” όπου στα κόκκινα φύλλα φτάνει λιγότερο φως στους χλωροπλάστες (ή/και λειτουργούν οι ανθοκυανίνες ως αντιοξειδωτικά), ενώ τα πράσινα φύλλα έχουν μεγαλύτερη δυνατότητα για μη φωτοχημική απόσβεση λόγω μεγαλύτερης συγκέντρωσης συστατικών του κύκλου των ξανθοφυλλών. Στο C. creticus, κατά την “πράσινη” περίοδο τα μελλοντικά ερυθρά φύλλα παρουσιάζουν φωτοσυνθετική και φωτοπροστατευτική κατωτερότητα, σε σχέση με τα φύλλα του πράσινου φαινοτύπου, που όμως δεν τους δημιουργεί πρόβλημα, όσο οι περιβαλλοντικές συνθήκες είναι ευνοϊκές. Κατά τη δυσμενή εποχή του χειμώνα, τα πράσινα φύλλα αυξάνουν την ικανότητα τους για μη φωτοχημική απόσβεση, ενώ τα κόκκινα όχι. Μπορεί σε αυτή την περίπτωση οι ανθοκυανίνες να αποτελούν μια προσαρμογή ώστε να αντισταθμίζεται αυτή η κατωτερότητα και να μειώνεται ο κίνδυνος φωτοαναστολής. Ωστόσο, τα νεαρά κόκκινα φύλλα του C. coccifera, είχαν υποστεί λιγότερες απώλειες από φυτοφάγα. Η μη ύπαρξη διακυμάνσεων στο φάσμα ανακλαστικότητας των κόκκινων φύλλων στην περιοχή 400-700 nm, σε συνδυασμό με τις δυνατότητες της χρωματικής όρασης πολλών φυτοφάγων εντόμων, υποδεικνύει ότι τα ερυθρά φύλλα πιθανόν να μην μπορούν εύκολα να εντοπιστούν από τους θηρευτές τους. Επίσης, κάποια φυλλοβόρα έντομα μπορεί να αποφεύγουν τα κόκκινα φύλλα, γιατί εκεί γίνονται περισσότερο ευδιάκριτα από τα αρπακτικά. Η αυξημένη συγκέντρωση των κόκκινων φύλλων σε φαινολικά μπορεί να αποθαρρύνει την περεταίρω κατανάλωση μετά από τυχαία προσέγγιση. / Leaves of some plants are transiently red due to anthocyanin accumulation. Among the many hypotheses for the function of foliar anthocyanins, two are tested in this field study: the sun screen, photoprotective function against excess visible light and the handicap signal against herbivory. Two plant species (Quercus coccifera and Cistus creticus) were used which display intraspecies variation in the expression of the anthocyanic character. Young leaves of some individuals of Q. cocifera are transiently red due to anthocyanin accumulation, while redness disappears upon maturation. Mature leaves of C. creticus are green during summer (“green” period) but in some individuals they turn transiently to red during winter (“red” period), while neighboring individuals remain green. In vivo chlorophyll fluorescence parameters, xanthophyll cycle pool sizes, reflectance spectra, total phenolics and extent of herbivory were compared in green and red leaves sampled from the corresponding phenotypes occupying the same habitat. An appreciable photosynthetic and photoprotective superiority (or inferiority) of anthocyanic leaves in both plant species under field conditions was not found. It seems that there is a compromise in differently coloured young leaves of Q. coccifera where red leaf chloroplasts enjoy less light due to its attenuation by anthocyanins (and/or anthocyanins participate in photoprotection through their anti-oxidant capacity) while green leaf chloroplasts are better equipped to dissipate excess light as heat through their higher size of xanthophyll cycle pool. During the “green” period, future red leaves of C. creticus show photosynthetic and photoprotective inferiority compared to the leaves of the green phenotype which is possibly insignificant for plant performance during the favourable period of the year. During the winter stress green leaves increase their ability for non-photochemical quenching while red phenotypes do not. In this case, anthocyanins may be an adaptation to compensate for this deficiency and alleviate the risk of photodamage. However, young red leaves of Q. coccifera are less attacked by insect consumers. The leveling of reflectance in anthocyanic leaves throughout the 400-570 nm band together with the spectral discriminating capabilities of leaf eating insects, may make red leaves less discernible to some insects. Furthermore, some insect herbivores may avoid red leaves because there are more discernible to predators. Incidental attack may be a posteriori discouraged due to the high phenolic investment of red leaves.