Isabel Fleck

Remodeling of tobacco thylakoids by over-expression of maize plastidial transglutaminase

Transglutaminases (TGases, EC 2.3.2.13) are intra- and extra-cellular enzymes that catalyze post-translational modification of proteins by establishing epsilon-(gamma-glutamyl) links and covalent conjugation of polyamines. In chloroplast it is well established that TGases specifically polyaminylate the light-harvesting antenna of Photosystem (PS) II (LHCII, CP29, CP26, CP24) and therefore a role in photosynthesis has been hypothesised (Della Mea et al. [23] and refs therein). However, the role of TGases in chloroplast is not yet fully understood. Here we report the effect of the over-expression of maize (Zea mays) chloroplast TGase in tobacco (Nicotiana tabacum var. Petit Havana) chloroplasts. The transglutaminase activity in over-expressers was increased 4 times in comparison to the wild-type tobacco plants, which in turn increased the thylakoid associated polyamines about 90%. Functional comparison between Wt tobacco and tgz over-expressers is shown in terms of fast fluorescence induction kinetics, non-photochemical quenching of the singlet excited state of chlorophyll a and antenna heterogeneity of PSII. Both in vivo probing and electron microscopy studies verified thylakoid remodeling. PSII antenna heterogeneity in vivo changes in the over-expressers to a great extent, with an increase of the centers located in grana-appressed regions (PSIIalpha) at the expense of centers located mainly in stroma thylakoids (PSIIbeta). A major increase in the granum size (i.e. increase of the number of stacked layers) with a concomitant decrease of stroma thylakoids is reported for the TGase over-expressers.

Leaf morphology, photochemistry and water status changes in resprouting Quercus ilex during drought

Functional and morphological (structural) characteristics of Quercus ilex L. leaves under drought stress were studied in the forest and in a nursery. We compared undisturbed individuals (controls) with resprouts emerging after clear-cut or excision. When soil water availability was high, gas-exchange was similar in resprouts and controls, despite higher midday leaf water potential, midday leaf hydration and relative water content (RWC). In moderate drought, stomatal closure was found to limit photosynthesis in controls, and in severe drought non-stomatal limitations of photosynthesis were also greater than in resprouts. Leaf structure and chemical composition changed under drought stress. Leaves tended to be smaller in controls with increasing drought, and resprouts had larger leaves and lower leaf mass area (LMA). The relationship between nitrogen (N) content and LMA implied lower N investment in photosynthetic components in controls, which could be responsible for their increased non-stomatal limitation of photosynthesis. Changes were more apparent in leaf density (D) and thickness (T), components of LMA. Decreases in D were related to reductions in cell wall components: hemicellulose, cellulose and lignin. In resprouts, reduced D and leaf T accounted for the higher mesophyll conductance (gmes) to CO2 measured.

Resource remobilization in Quercus ilex L. resprouts

We studied the source of the nitrogen used for the growth and resprouting of holm-oak (Quercus ilexL.), and the contribution of nitrogen and carbohydrate root reserves to these processes. Three-year-old plants were grown in a greenhouse with either a sufficient or restricted nitrogen supply for one year. Half the individuals were subjected to shoot excision to provoke resprouting, and a 15N solution was given to these plants and to controls for two months. Nitrogen, Total Non-structural Carbohydrate (TNC), Total Soluble Protein content, and 15N and 13C composition were determined, and histological analyses of woody tissue were performed. Our results show that N-deprived plants used nitrogen from root reserves to support a growth rate similar to that of non-deprived plants. However, deprived plants lost their resprouting capacity in spite of the high TNC accumulation and nitrogen resupply to the soil. After the supply of nitrogen was restored to N-deprived plants, this nutrient mainly accumulated in under-ground organs, which limited the above-ground growth. Resprouting plants first remobilized the nitrogen stored in roots, and thereafter took it up from the solution. The root-crown region did not behave as a specialised reserve organ in three-year-old Quercus ilex L. plants.

Carbon isotope discrimination in Quercus ilex resprouts after fire and tree-fell

Ecophysiological differences related to photosynthesis were compared in holm oak Quercus ilex leaves from undisturbed holm-oak vegetation, resprouts after fire and resprouts after tree-fell. No significant differences in any parameter measured were observed between the two kinds of resprout throughout the first growing season following disturbance. Resprouting leaves showed lower carbon isotope discrimination (Δ) and intercellular CO2 concentration (pi), and higher photosynthesis, leaf conductance and transpiration rates than leaves from undisturbed stands. Nitrogen, soluble protein content and ribulose bisphosphate carboxylase (RuBPCase) activity were 88%, 96% and 45% higher respectively, in both kinds of resprout. The results indicate that photosynthetic capacity, rather than stomatal conductance, is the limiting factor in photosynthesis in resprouts, Chlorophyll content and chlorophyll a/b ratio did not differ between resprouts and undisturbed leaves, indicating that the observed differences were not a result of differences in light environment during leaf development. Leaf mass per area (LMA), was 80% higher in the resprouts, and was negatively related (r=−0.86) to Δ and positively related (r=0.87) to N content. Enhanced carbon assimilation after disturbances resulted in higher water use efficiency, as indicated by lower Δ values in the resprouts. We conclude that the cause of defoliation was not relevant in the physiology of the resprouts, suggesting the importance of underground organs.

Variations in Quercus ilex chloroplast pigment content during summer stress: involvement in photoprotection according to Principal Component Analysis

We examined chloroplast pigment variation in holm oak (Quercus ilex L.) leaves for two periods under two climatic conditions, at midday during summer. We compared variation between control (unburned) plants and plants burned the preceding summer, since post-fire resprouts show higher photosynthetic rates and lower thermal energy dissipation. Principal component (PC) analysis was performed on nine pigment-content variables for the two periods separately. Two PC factors (PC1 and PC2) explained 83 and 84% of the variance of the data for each period. In both periods, PC1 was marked by positive loading of pigments associated with light absorption or structural function namely neoxanthin, lutein, β-carotene, chlorophyll a, and chlorophyll b. These pigments were only affected by leaf age. In contrast, PC2 was marked by high loadings of xanthophyll-cycle pigments (associated with photoprotection), and lutein-5,6-epoxide. Leaf content of these pigments was affected by climatic conditions. In the situations considered in PC analysis (leaf types, periods), the lutein-5,6-epoxide content presented a variation pattern similar to that of violaxanthin, and was significantly correlated with thermal dissipation of excess energy (represented by non-photochemical quenching or NPQ). These results suggest a relationship of lutein and lutein-5,6-epoxide with photoprotection.

Leaf flavonoid content in Quercus ilex L. resprouts and its seasonal variation

Here, we provide the first report on flavonoid content in holm oak (Quercus ilex L.) leaves, analyzed by HPLC–MS/MS. Flavanols and flavonols were the predominant groups, although proanthocyanidins and many soluble tannins had a relevant presence in all leaf samples. Seasonal variation of flavonoids was determined in extracts from Q. ilex leaves during resprouting after a forest fire in two Mediterranean forests. Similar seasonal trends were observed over 2 years during the two main stress seasons (winter and summer). The most abundant flavonoid was the flavanol epicatechin, which showed similar values during the two seasons. Hexosides of the flavonols, quercetin, kaempferol and rhamnetin showed considerably higher content in winter, especially at the lowest temperatures. These variations in both forests are discussed on the basis of the chlorophyll fluorescence results obtained. Anthocyanins were found practically absent in mature leaves. Nutrient or water availability differences between sites or seasons were not related to changes in leaf flavonol-hexoside content.

Total antioxidant activity in Quercus ilex resprouts after fire

Abstract After fire, holm oak ( Quercus ilex L.) resprouts have a higher light availability and photosynthetic activity than control plants in intact vegetation. To assess the differences in protection between these plants, we determined, in two forests, changes in gas-exchange rates, chlorophyll fluorescence parameters, chloroplast pigment content and total antioxidant activity (TAA) in different seasons, at different times of the day and in relation to leaf age. The end-point method used for TAA determination allowed the evaluation of the relative contribution of hydrophilic and lipophilic antioxidants in leaf extracts. High correlations were obtained between lipophilic TAA and certain chloroplast pigment content, the highest contribution being provided by β-carotene and the components of the xanthophyll cycle (zeaxanthin + antheraxanthin, violaxanthin). All leaves showed a much higher contribution (94–99%) of hydrophilic than lipophilic antioxidants to TAA. In summer, at midday, photosynthetic rates and TAA were higher in resprouts. In these plants, reduced xanthophyll-cycle participation (as shown by non-photochemical quenching (NPQ) and xanthophyll pool content) was compensated by an increased participation of hydrophilic antioxidants. No effect of time of day or plant age was observed. The results suggest that energy dissipation as heat, and detoxification mechanisms contribute to the protective strategies of control plants and resprouts, albeit to a different extent.

Oxidative stress induced in tobacco leaves by chloroplast over-expression of maize plastidial transglutaminase

As part of a project aiming to characterize the role of maize plastidial transglutaminase (chlTGZ) in the plant chloroplast, this paper presents results on stress induced by continuous chlTGZ over-expression in transplastomic tobacco leaves. Thylakoid remodelling induced by chlTGZ over-expression in young leaves of tobacco chloroplasts has already been reported (Ioannidis et al. in Biochem Biophys Acta 1787:1215–1222, 2009). In the present work, we determined the induced alterations in the photosynthetic apparatus, in the chloroplast ultrastructure, and, particularly, the activation of oxidative and antioxidative metabolism pathways, regarding ageing and functionality of the tobacco transformed plants. The results revealed that photochemistry impairment and oxidative stress increased with transplastomic leaf age. The decrease in pigment levels in the transformed leaves was accompanied by an increase in H2O2 and lipid peroxidation. The rise in H2O2 correlated with a decrease in catalase activity, whereas there was an increase in peroxidase activity. In addition, chlTGZ over-expression lead to a drop in reduced glutathione, while Fe-superoxide dismutase activity was higher in transformed than in wild-type leaves. Together with the induced oxidative stress, the over-expressed chlTGZ protein accumulated progressively in chloroplast inclusion bodies. These traits were accompanied by thylakoid scattering, membrane degradation and reduction of thylakoid interconnections. Consequently, the electron transport between photosystems decrease in the old leaves. In spite of these alterations, transplastomic plants can be maintained and reproduced in vitro. These results are discussed in line with chlTGZ involvement in chloroplast functionality.

Mesophyll conductance to CO2 and leaf morphological characteristics under drought stress during Quercus ilex L. resprouting

Abstract• Quercus ilex L., the dominant species in Mediterranean forests and one with a great capacity for resprouting after disturbances, is threatened by the expected increase in fire frequency and drought associated with climate change.• The aim of this study was to determine the contribution of photosynthesis limitants, especially mesophyll conductance (gmes) during this species’ resprouting and under summer drought.• Resprouts showed 5.3-fold increased gmes and 3.8-fold increased stomatal conductance (gs) at mid-day with respect to leaves of undisturbed individuals. With increased drought, structural changes (decreased density and increased thickness) in resprouts contributed to the observed higher photosynthesis and increased gmes. However, gmes only partially depended on leaf structure, and was also under physiological control. Resprouts also showed lower non-stomatal limitations (around 50% higher carboxylation velocity (Vc,max) and capacity for ribulose-1,5-bisphosphate regeneration (Jmax)). A significant contribution of gmes to leaf carbon isotope discrimination values was observed.• gmes exhibits a dominant role in photosynthesis limitation in Q. ilex and is regulated by factors other than morphology. During resprouting after disturbances, greater capacity to withstand drought, as evidenced by higher gmes, gs and lower non-stomatal limitants, enables increased photosynthesis and rapid growth.Résumé• Quercus ilex L., l’espèce dominante dans les forêts méditerranéennes qui a une grande capacité de rejets après des perturbations, est menacée par l’augmentation prévue de la fréquence des incendies et de la sécheresse associées au changement climatique.• Le but de cette étude était de déterminer, chez cette espèce, la contribution des limitations de la photosynthèse, en particulier de la conductance du mésophylle (gmes) au cours de la repousse et sous sécheresse estivale.• Les feuilles des rejets ont présenté une conductance mésophylienne (gmes) 5,3 fois plus élevée et une conductance stomatique (gs) à midi 3,8 fois plus élevée par rapport aux feuilles d’arbres non perturbés. Avec l’accroissement de la sécheresse, les changements de structures (diminution de la densité et épaisseur accrue) dans les rejets ont contribué à augmenter la photosynthèse et à accroître gmes. Toutefois, gmes dépendait partiellement de la structure des feuilles, et était également sous contrôle physiologique. Les rejets ont aussi montré une abscence de limitation stomatique (vitesse de carboxylation (Vc,max) environ 50% plus élevée et une capacité de régénération pour le ribulose-1,5-bisphosphate (Jmax). Une contribution significative de gmes à la discrimination isotopique du carbone dans les feuille a été observée.• La conductance mésophylienne (gmes) a présenté un rôle dominant dans la limitation de la photosynthèse chez Q. ilex et est régulée par des facteurs autres que la morphologie. Au cours de la repousse après des perturbations, une plus grande capacité à résister à la sécheresse, mise en évidence par une gmes et une gs plus élevées, et une diminution des limitations non stomatiques, permettent une augmentation de la photosynthèse et une croissance rapide.

Response of transglutaminase activity and bound putrescine to changes in light intensity under natural or controlled conditions in Quercus ilex leaves

In order to further study a previously observed relationship between polyamine (PA) content and changes in irradiation, we examined the level of free and bound PAs, the activity of transglutaminase (TGase, EC 2.3.2.13) and chlorophyll fluorescence in holm oak (Quercus ilex L.) leaves in response to different levels of light intensity and amount. A diurnal trend of free and bound putrescine (F-Put and B-Put, respectively) and TGase activity was observed in plants under natural conditions in the forest, with the highest value corresponding to the maximum light intensity and amount of light received by the leaves. In another set of experiments, potted Q. ilex plants in experimental fields were subjected to a range of periods of natural photosynthetic photon flux density (PPFD) by covering or not covering the whole trees. Under a natural photoperiod (uncovered leaves), B-Put content and TGase activity paralleled the diurnal PPFD pattern, reaching a maximum at the highest PPFD; prior to this maximum, free PAs showed a significant rise. Plants that were in darkness until midday and suddenly exposed to high light intensity showed enhanced TGase activity, resulting in the maximum accumulation of B-Put. The involvement of the accumulation of B-Put reflected in the changes of the B-Put/bound spermidine ratio during the photoprotective responses to high light stress in forest plants is discussed in relation to the chlorophyll fluorescence parameters observed.