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Photosynthetic organisms use sunlight as the primary source of energy to support their metabolism. In eukaryotes, reactions responsible of the conversion of light into chemical energy occur in specific organelles, the chloroplasts. In this study, we showed that mitochondria also have a seminal influence on cells' energy metabolism and on photosynthetic reactions. This is illustrated by the observation that the strong photosensitivity of

Oxygenic photosynthesis is a crucial process for life on earth as it enables plants and algae to convert sunlight into chemical energy, generating molecular oxygen as a byproduct. Light can also be harmful and when in excess can drive to photosystems over‐excitation and production of reactive oxygen species (ROS) with the consequent decrease of the overall photosynthetic efficiency. In a highly dynamic natural environment photosynthetic organisms have evolved sophisticated mechanisms to modulate their efficiency to capture and exploit light. For instance the so called non photochemical quenching of fluorescence (NPQ) acts dissipating excess energy as heat and it’s used as short term response to high light in order to avoid oxidative damages. The carotenoid zeaxanthin belonging to the xanthophyll cycle enhances this thermal dissipation but also has a direct role in the scavenging of ROS generated in the membrane. Acclimation instead is a more complex long term process that acts directly modeling the composition of the photosynthetic apparatus in response to different light intensity, for example through modifications in protein composition. Photoregulation and photoprotection are strongly related also to modulations of flow of excitation energy and electrons across the thylakoid membrane. Indeed the major pathway for the light reactions of photosynthesis, the linear electron flow, can modulate its rate depending on metabolic demand and can be also supported by alternative electron pathway which affect the thylakoid gradient across the membrane and the ATP/NADPH ratio. The general aim of this work is to investigate the different mechanisms modulating photosynthetic efficiency in the microalga Nannochloropsis gaditana in order to increase the limited knowledge about this interesting microalga and exploit it to optimize photosynthetic efficiency in a large-scale cultivation perspective, even through the development of computational models. The spectroscopic tools developed to untangle the complexity of the photosynthetic regulation in Nannochloropsis have been successfully applied to study photosynthesis in other photosynthetic organisms such as, Chlamydomonas reinhardtii, Physcomitrella patens and Koliella antarctica. Nannochloropsis gaditana is an eukaryotic alga of the phylum of heterokonta, originating from a secondary endosymbiotic event. Species of this group have received increasing attention in the scientific community, reflecting their potential application in biofuel production, although the photosynthetic and physiological properties of these organisms remain poorly characterized. Nannochloropsis species have a peculiar photosynthetic apparatus characterized by the presence of chlorophyll a, violaxanthin and vaucheriaxanthin as the most abundant pigments. The regulation of the photosynthetic apparatus in this interesting microalgae has been deeply discussed in Section B. Our study focused firstly on the acclimation response in Nannochloropsis gaditana subjected to prolonged exposition to low and high light. Intense illumination induces a decrease in the chlorophyll content and the antenna size of both Photosystem I and II. Cells grown in high light also show increased photosynthetic electron transport, paralleled by an increased contribution of cyclic electron transport around Photosystem I. Even when exposed to extreme light intensities, Nannochloropsis cells do not activate photo-protection responses, such as NPQ and the xanthophyll cycle in a constitutive way. Conversely, these responses remained available for activation upon additional changes in illumination. These results suggest NPQ and the xanthophyll cycle in Nannochloropsis gaditana play exclusive roles in response to short-term changes in illumination but only play a slight role, if any, in responses to chronic light stress. In order to further explore the short term response mediated by xanthophyll cycle the effect of zeaxanthin accumulation in the photosynthetic apparatus of Nannochloropsis gaditana was investigated revealing some peculiar aspects. Interestingly zeaxanthin molecules are found to be constitutively present in this microalga, even in conditions of very low light in which the xanthophyll cycle is not yet induced. In addition this xanthophyll does not show a specific binding site in the different protein components of the photosynthetic apparatus and, in addition, has a strong effect in the NPQ response. The influence on NPQ seems to be related mostly on de novo synthesis of zeaxanthin while the molecules already present in the photosynthetic apparatus are involved in a transient NPQ active only in the first minute after the dark-light transition. The regulation of the photosynthetic apparatus has been assessed also in N. salina in a growing system more compatible with a large-scale production system, a continuous-flow flat-plate photobioreactor. Interestingly changing the residence time maintaining the same irradiation affects the biomass concentration leading to an acclimation response very similar to that observed for N. gaditana grown in batch system, as previously discussed. These results highlight the importance of the biomass concentration and its connection with light supply as parameter to optimize in order to increase the microalgal culture productivity. The molecular investigation of the mechanisms at the basis of light exploitations in Nannochloropsis is the starting point for the development of computational models that aim to simulate and predict microalgae behavior in order to optimize their productivity in large-scale cultivation systems. Section C deals with the development and widespread application of these models, which integrate chlorophyll fluorescence measurements allowing also the representation of complex mechanisms such as NPQ. Such models prove especially useful in identifying which parameters have the largest impact on productivity, thereby providing a means for enhancing growth through design and operational changes. They can also provide guidance for genetic engineering by identifying those modifications having the largest potential impact on productivity. In Section D the study of photosynthetic processes is expanded to other organisms focusing on the regulation of the photosynthetic electron chain through the employment of several spectroscopic approaches set up during my PhD thesis. In the first work reported we show that the introduction of a mitochondrial mutation in Chlamydomonas reinhardtii mutants depleted in the chloroplastic PGRL1 rescue its photosensitivity in high light. Detailed functional analysis of these cells showed that the mitochondria mutation alters the electron transport reactions increasing alternative electron pathways around PSI at the detriment of PSII-related photosynthesis. This work thus clearly shows how mitochondrial activity play a seminal influence on photosynthesis in algae. The second work presented deals with another important mechanisms to modulate flow of excitation, the Mehler-like reactions mediated by Flavodiiron (FLV) proteins. These proteins were lost during evolution of land plants but are still present in non vascular plants, as the moss Physcomitrella patens, the model organism employed for this study. P. patens mutants depleted in FLV show these proteins are active as an electron sink downstream of Photosystem I. Measurement of electron transport showed that they play a major role particularly in the first seconds after a sudden change in light intensity, when for a few seconds they are the major sink for electrons from PSI. When exposed to fluctuating light FLV mutants showed light sensitivity and PSI photoinhibition, demonstrating their biological role as a safety valve for excess electrons in dynamic light. FLV absence in mutants was, in part, compensated by increased cyclic electron flow, suggesting that their biological role may have been substituted in vascular plants by this other mechanism of alternative electron flow. Finally we analyzed the time course of physiological and morphological responses to different irradiances in Koliella antarctica, a green antarctic microalga isolated from Ross Sea. K. antarctica not only modulates cell morphology and composition of its photosynthetic apparatus on a long-term acclimation, but also shows the ability of a very fast response to light fluctuations. The ability to activate such responses is fundamental for survival in its natural extreme environment.

Additional file 1. Table S1. In vivo Chl fluorescence parameters used for screening mutant strains. Figure S1. Verification of the successful integration of the Sh-ble gene in N. gaditana colonies. Figure S2. Example of fluorescence screening using homogenized inoculum. Figure S3. Outline of the three-step screening used to isolate N. gaditana photosynthetic mutant strains. Figure S4. Growth rates and pigment contents of selected insertional mutant strains. Figure S5. Western blot analysis of strains with reduced Chl contents that were selected from both screens.

Additional file 1. Table S1. In vivo Chl fluorescence parameters used for screening mutant strains. Figure S1. Verification of the successful integration of the Sh-ble gene in N. gaditana colonies. Figure S2. Example of fluorescence screening using homogenized inoculum. Figure S3. Outline of the three-step screening used to isolate N. gaditana photosynthetic mutant strains. Figure S4. Growth rates and pigment contents of selected insertional mutant strains. Figure S5. Western blot analysis of strains with reduced Chl contents that were selected from both screens.