Diploma thesis’s aim was to determine Rubisco activity and its content in sunny and shaded needles of Norway spruce (Picea abies [L.] Karst.) and confirm or refuse acclimation on Rubisco enzyme level. Samples were taken on the Experimental ecology station Czech Academy of Sciences at Bílý Kříž (Beskydy Mountains) where needles were cultivated in two cultivation spheres with different concetrations CO2. One cultivation sphere contains atmosphere with ambient (A) concentration CO2 (350 mol CO2 mol-1) and the other contains atmosphere with elevated (E) concentration CO2 (700 mol CO2 mol-1). Rubisco activities were determinated spectrophotometric, Rubisco content was determined by SDS-PAGE method. Samples were taken in the modele of June and in the end of September. Elevated concetration CO2 did not have significant influence on Rubisco activity. Whereas different exposition had significant influence on Rubisco activity in June, when was found lower Rubisco activity in shaded needles than in sunny needles. In September were not find significant differences in Rubisco activity beetwen sunny and shaded needles. We did not find statistically significant differences in Rubisco content between A and E needles in June. Incontrary, we found about 34 % lower Rubisco content in E needles than in A needles, which confirms acclimation on Rubisco enzyme level. In September Rubisco content was several time higher than in June, which evidences the long term building photosynthetic apparatus in coniferous trees.

This diploma thesis examines influence of content of leaf nitrogen and non-structural carbohydrates on Rubisco activity and its content under impact of ambient (A = 385 µmolCO2 mol-1) and elevated (E = 700 µmolCO2 mol-1) concentrations of carbon dioxide in needles of Norway spruce (Picea abies L.). The Rubisco activity was determined spectrophotometrically, the Rubisco content by using SDS-PAGE, the content of non-structural carbohydrates by using the anthron method and total leaf nitrogen was determined by using the Kjeldahl method. The Rubisco activity in needles of Norway spruce did not show acclimation on elevated concentration of carbon dioxide, but the activity was elevated concentration of carbon dioxide stimulated during the growing season. The acclimation was proved by Rubisco content, its content was significantly lower in needles cultivated under elevated carbon dioxide. Content of total leaf nitrogen was statistically significantly lower in needles of Norway spruce cultivated under elevated carbon dioxide then in needles cultivated under ambient concentration of carbon dioxide. On the contrary, the content of non-structural carbohydrates was significantly higher in needles cultivated under elevated concentration of carbon dioxide then in variant A. Seasonal changes of Rubisco content depended on content of non-structural carbohydrates, the Rubisco content decreased with increasing content of non-structural carbohydrates. No dependence was detected between seasonal changes of the total leaf content and the Rubisco content.

Kolavskiljning, avlägsnande av koldioxid (CO2) från atmosfären, har fått uppmärksamhet som en metod för att mildra effekterna av den globala uppvärmningen. Växter och fototrofa mikroorganismer har den inneboende förmåganatt fånga upp kol genom fixering av CO2 för att producera biomassa. Däremot inhemska kolfixeringsvägar begränsas av nyckelenzymer med låg katalytisk aktivitet vilket resulterar i låg energieffektivitet. Rubisco är en sådan nyckelenzym, ökänt för sin dåliga prestanda. Tidigare forskning har misslyckats när det gäller att förbättra kolet fixering i Rubisco med konventionella metoder. Generativ modellering har dykt upp som en innovativ förhållningssätt till enzymteknik, dra fördel av olika arkitekturer för neurala nätverk för att föreslå en ny varianter med önskade egenskaper. Här tränas en variationsautokodare (VAE) på Rubisco-sekvensen utrymme användes för utmaningen med Rubiscos ingenjörskonst. Två modeller utbildades och med hjälp av dimensionsreduktionsegenskapen hos VAE, utforskades fitnesslandskapet i Rubisco. Sekvenser var märkt med katalytiskt relevanta data och en regressionsmodell byggdes med syftet att förutsäga dessa sekvenser med ökad katalytisk aktivitet. Nya Rubisco-sekvenser genererades efter systematiska utfrågning av det lågdimensionella rummet. Användningen av generativ modellering här ger ett nytt perspektiv på Rubisco engineering. / Carbon capture, the removal of carbon dioxide (CO2) from the atmosphere, has gained attention as a method to mitigate the effects of global warming. Plants and phototrophic microorganisms have the inherent ability to capture carbon through the fixation of CO2 to produce biomass. However, native carbon fixing pathways are limited by key enzymes with low catalytic activity resulting in low energy efficiency. Rubisco is one such key enzyme, notorious for its poor performance. Past research has been unsuccessful at enhancing carbon fixation in Rubisco through conventional methods. Generative modelling has emerged as an innovative approach to enzyme engineering, taking advantage of different neural network architectures to propose novel variants with desired characteristics. Here, a variational autoencoder (VAE) trained on the Rubisco sequence space was applied to the challenge of Rubisco engineering. Two models were trained and, using the dimensionality reduction property of VAEs, the fitness landscape of Rubisco was explored. Sequences were labelled with catalytically relevant data and a regression model was built with the aim of predicting those sequences with enhanced catalytic activity. Novel Rubisco sequences were generated following systematic interrogation of the low-dimensional space. The use of generative modelling here provides a fresh perspective on Rubisco engineering.

Photosynthesis is responsible for fixing approximately 111 – 117 Pg of CO₂ into organic carbon each year, of which about half is performed by algae in the oceans. Over geological timescales, photosynthesis by algae was instrumental in transforming Earth’s atmosphere. Despite the integral role algae play in the carbon cycle, the interaction and feedbacks between CO₂ fixation by algae and atmospheric CO₂ is poorly understood. This thesis expands upon our current knowledge by tracing the evolution of the key enzyme of photosynthesis, Rubisco, in algae through geological history. It was found that Rubisco underwent adaptation during distinct periods corresponding with falling atmospheric CO₂. The pattern of adaptation hints at physiological adaptation to varying concentrations of atmospheric CO2 and possibly indicates the emergence of carbon concentrating mechanisms (CCMs). This adaptation was probed further within the red and chromist algae, identifying key residues within the Rubisco protein sequence that may influence its kinetic properties. This research also provided new measurements of Rubisco CO2 affinity within the haptophyte algae. Finally, the importance of HCO₃- use by phytoplankton in the modern ocean was explored. HCO₃- utilisation was modelled through signals retained within stable carbon isotopes of organic matter estimate the response to anthropogenic increases of CO₂. The results indicate that phytoplankton utilise a large proportion HCO₃- which shows little sensitivity to anthropogenic increases of CO₂, even when model predictions are extended to 2100. This thesis demonstrates how algae can respond to CO₂ levels over geological and anthropogenic time scales.

The atmospheric CO2 concentration is rising. These higher CO2 concentrations will certainly affect growth but may also affect nutrient responses of crop plants. The overall aim of this thesis was to investigate the effect of elevated CO2 on the nitrogen and phosphorus nutrition of two different crop species: wheat and cotton. Once establishing the new nutrient requirements of these crop species at elevated CO2 the study focussed on the interactive effects of elevated CO2 and N supply on growth and leaf N concentrations in cotton and wheat, carbohydrate metabolism, source:sink interactions and photosynthetic enzyme levels in the two species. Having established these effects, the role of N supply in controlling source:sink effects on Rubisco concentration were studied at the molecular level in wheat. Finally, some implications of the effect of elevated CO2 on wheat were examined, specifically on the quality of flour produced from wheat grain grown at elevated CO2. The foliar nitrogen concentrations, required to produce maximum shoot growth (critical concentrations) were lower at elevated CO2 while the critical phosphorus concentrations were higher. / Doctor of Philosophy (PhD)

Symbiosis is commonly present in marine invertebrates. Many corals and sponges have symbiotic algae or bacteria. In the previous studies of the sponge Cinachyra australiensis, 85% of the bacteria associated with the sponge have high similarity (88.65%) with the symbiotic chemoautotrophic sulfur-oxidizing bacteria of the deep-sea hydrothermal vent mussel, Solemya reidi. This study aims to investigate the localization of the chemoautotrophic sulfur-oxidizing bacteria associated with Cinachyra australiensis. The Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) large-subunit genes for autotrophic organisms were amplified by polymerase chain reaction from the sponge samples. The phylogenetic relationship of the RubisCO large subunit genes was analyzed. A total of 26 clones were selected and sequenced. They could be divided into two groups. One (9 clones) belongs to form I type IB (cynobacteria and green algae). The other (17 clones) belongs to form II type IA (chemoautotrophic symbiotic bacteria). The location of the sulfur-oxidizing chemoautotrophic bacteria was shown to be intracellular symbiosis within the mesoglial cells by fluorescence in situ hybridization.

Salinity usually reduces plant growth in terms of height and biomass, but can increase secondary metabolite production. This frequently reported observation in guayule (Parthenium argentatum Gray, Asteraceae) was investigated for possible mechanisms.Osmotic and specific ion effects of four chloride salts (CaCl2, MgCl2, KCl, and NaCl) on leaf anatomical and plant physiological parameters were studied. One-year-old plants of guayule line AZ 2 were grown under two salt concentrations (750 ppm and 1500 ppm) for each salt type (plus a control) in sand culture (semi-hydroponic) for eight weeks under controlled greenhouse conditions in Tucson, Arizona.Growth in height decreased with increasing salt concentration. Shoot dry weight, rubber, and resin contents, however, showed no significant differences between treatments, indicating no effect from either salt concentration or salt type. There was a trend for increasing rubber content with increasing salt concentration, although not statistically significant. At the same time, net CO2 gas exchange rates decreased significantly with increasing salinity.With increasing salt concentration, guayule showed osmotic effects in terms of height, indicating a lower hydraulic conductivity. Although plants of higher salt concentrations utilized significantly less water, they had the same shoot dry weights, rubber, and resin contents. Salt-stressed plants therefore achieved higher water use efficiencies. The diurnal net CO2 gas exchange rates were significantly reduced with increasing salinity; the nocturnal net CO2 gas exchange rates showed no significant difference between the treatments.Anatomically, it was found that the stomata were raised or elevated above the epidermis, and supported by upwardly curving cells. When guayule was grown under salt treatments, the trichomes were found to include deposits of material. Trichomes might act as a detoxification repository for excess ions. Although the physiological significance of raised stomata is unknown, it is hypothesized that the unique combination of raised stomata, indumentum, and multiple layers of palisade parenchyma allows for an overall high photosynthetic capacity and performance. During stress conditions such as salinity or drought, guayule might activate an internal CO2 concentrating mechanism, i.e., bicarbonate/CO2 pump, internal CO2 recycling, or PEP carboxylation activity.

Verticillium is a soil-borne fungus that is one of the world's foremost plant pathogens. Commercial plant grafting suggests that resistance occurs in the root; this conflicts with decades of research indicating that resistance occurs in the stem. The goal of this thesis work was to use an alternative approach to determine the location of resistance by expressing the Ve1 gene using organ-specific promoters. Promoter sequences for the stem-specific gene, Ribulose 1,5-bisphosphate carboxylase oxygenase small chain 2A (Rbsc2A), and root-specific gene, Tobacco Mosaic Virus Induced (TMVi) were taken from the Sol Genomics Network (SGN) database, cloned into constructs with the Ve1 gene and susceptible tomato germplasm was transformed using Agrobacterium tumefaciens. Preliminary results suggest that resistance may not be localized and expression of the Ve1 gene in either the root or the stem is sufficient to develop whole plant resistance to the Verticillium pathogen.

Five different forms of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO; IA, IB, IC, ID, II), the carboxylase of the Calvin-Benson-Bassham cycle (CBB), are utilized by plants, algae and autotrophic bacteria for carbon fixation. Discrimination against 13C by RubisCO is a major factor dictating the stable carbon isotopic composition (δ13C = {[13C/12C sample/13C/12C standard] - 1} X 1000) of biomass. To date, isotope discrimination, expressed as ε values (={[12k/13k] - 1} X 1000; 12k and 13k = rates of 12C and 13C fixation) has been measured for form IA, IB, and II RubisCOs from only a few species, with ε values ranging from 18 to 29 /. The aim of this study was to better characterize form ID and IC RubisCO enzymes, which differ substantially in primary structure from the IB enzymes present in many cyanobacteria and organisms with green plastids, by measuring isotopic discrimination and kinetic parameters (KCO2 and Vmax). Several major oceanic primary producers, including diatoms, coccolithophores, and some dinoflagellates have form ID RubisCO, while form IC RubisCO is present in many proteobacteria of ecological interest, including marine manganese-oxidizing bacteria, some nitrifying and nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as Rhodobacter sphaeroides. The ε - values of the form ID RubisCO from the coccolithophore, Emiliania huxleyi and the diatom, Skeletonema costatum (respectively 11.1 / and 18.5 /) were measured along with form IC RubisCO from Rhodobacter sphaeroides and Ralstonia eutropha (respectively 22.9 / and 19.0 /). Isotopic discrimination by these form ID/IC RubisCOs is low when compared to form IA/IB RubisCOs (22-29 /). Since the measured form ID RubisCOs are less selective against 13C, oceanic carbon cycle models based on 13C values may need to be reevaluated to accommodate lower ε values of RubisCOs found in major marine algae. Additionally, with further isotopic studies, the extent to which form IC RubisCO from soil microorganisms contributes to the terrestrial carbon sink may also be determined.

Changing climate in combination with growing world populations mean that there is growing need for plants to be grown on land that is currently considered marginal for agriculture. Sorghum is a C4 plant that serves as an important food crop in Africa and India. It is also known to be highly drought tolerant but the mechanisms responsible for this tolerance are unclear. The overall aim of this study was to understand the drought tolerance mechanisms that enable the plant to maintain leaf function for a long time during water deficit. In Chapter 2 of this thesis, I studied the underlying physiological mechanisms for tolerating drought in two sorghum varieties with differing degrees of drought tolerance compared to a closely related species, Zea mays. During progressive drought, the more tolerant sorghum variety Samsorg 17 maintained net CO2 assimilation and photochemistry longest relative to the less tolerant Samsorg 40 and Zea mays. Differences were also seen in stomatal aperture, stomatal density, total chlorophyll content, chl a:b and A/Ci curve responses with maize more affected than the sorghum varieties. In Chapter 3, I identified novel drought tolerance mechanisms in the sorghum varieties. The less tolerant Samsorg 40 lost PsbA (D1) and Rubisco proteins and reengineered its photosynthetic apparatus to accumulate amino acids and sugars in order to maximise survival under drought. Samsorg 17 maintained photosynthetic proteins notably PsbA (D1) and Rubisco and accumulated high constitutive sugar content allowing for the maintenance of transpiration and photosynthesis. The two sorghum varieties had strikingly contrasting approaches of tolerating drought as demonstrated in Chapter 3. In Chapter 4, the aim was to characterise biochemical and metabolic changes that occur in response to drought. In particular, to identify sugars that are accumulated constitutively in Samsorg 17 and nitrogen sinks for lost N in Samsorg 40. My findings indicated a contrasting response in terms of sugar content in Samsorg 17 but support for amino acids as N sinks in Samsorg 40 as reported earlier. Sugars, sugar alcohols, lipids, organic acids, heat shock proteins and dehydrins were generally higher or more induced in Samsorg 17 relative to Samsorg 40. Samsorg 40 rather made amino acids. The implications of my findings and future work arising from this study were discussed in detail in the final chapter. In conclusion, in this thesis, it was demonstrated that closely related plants can have mechanistically different physiological and biochemical mechanisms for responding to drought.