Mario Giordano

Sulfur Assimilation in Photosynthetic Organisms: Molecular Functions and Regulations of Transporters and Assimilatory Enzymes

Sulfur is required for growth of all organisms and is present in a wide variety of metabolites having distinctive biological functions. Sulfur is cycled in ecosystems in nature where conversion of sulfate to organic sulfur compounds is primarily dependent on sulfate uptake and reduction pathways in photosynthetic organisms and microorganisms. In vascular plant species, transport proteins and enzymes in this pathway are functionally diversified to have distinct biochemical properties in specific cellular and subcellular compartments. Recent findings indicate regulatory processes of sulfate transport and metabolism are tightly connected through several modes of transcriptional and posttranscriptional mechanisms. This review provides up-to-date knowledge in functions and regulations of sulfur assimilation in plants and algae, focusing on sulfate transport systems and metabolic pathways for sulfate reduction and synthesis of downstream metabolites with diverse biological functions.

Algal evolution in relation to atmospheric CO2: carboxylases, carbon-concentrating mechanisms and carbon oxidation cycles

Oxygenic photosynthesis evolved at least 2.4 Ga; all oxygenic organisms use the ribulose bisphosphate carboxylase-oxygenase (Rubisco)–photosynthetic carbon reduction cycle (PCRC) rather than one of the five other known pathways of autotrophic CO2 assimilation. The high CO2 and (initially) O2-free conditions permitted the use of a Rubisco with a high maximum specific reaction rate. As CO2 decreased and O2 increased, Rubisco oxygenase activity increased and 2-phosphoglycolate was produced, with the evolution of pathways recycling this inhibitory product to sugar phosphates. Changed atmospheric composition also selected for Rubiscos with higher CO2 affinity and CO2/O2 selectivity correlated with decreased CO2-saturated catalytic capacity and/or for CO2-concentrating mechanisms (CCMs). These changes increase the energy, nitrogen, phosphorus, iron, zinc and manganese cost of producing and operating Rubisco–PCRC, while biosphere oxygenation decreased the availability of nitrogen, phosphorus and iron. The majority of algae today have CCMs; the timing of their origins is unclear. If CCMs evolved in a low-CO2 episode followed by one or more lengthy high-CO2 episodes, CCM retention could involve a combination of environmental factors known to favour CCM retention in extant organisms that also occur in a warmer high-CO2 ocean. More investigations, including studies of genetic adaptation, are needed.

Algal and aquatic plant carbon concentrating mechanisms in relation to environmental change.

Carbon dioxide concentrating mechanisms (also known as inorganic carbon concentrating mechanisms; both abbreviated as CCMs) presumably evolved under conditions of low CO2 availability. However, the timing of their origin is unclear since there are no sound estimates from molecular clocks, and even if there were, there are no proxies for the functioning of CCMs. Accordingly, we cannot use previous episodes of high CO2 (e.g. the Palaeocene–Eocene Thermal Maximum) to indicate how organisms with CCMs responded. Present and predicted environmental change in terms of increased CO2 and temperature are leading to increased CO2 and HCO3− and decreased CO32− and pH in surface seawater, as well as decreasing the depth of the upper mixed layer and increasing the degree of isolation of this layer with respect to nutrient flux from deeper waters. The outcome of these forcing factors is to increase the availability of inorganic carbon, photosynthetic active radiation (PAR) and ultraviolet B radiation (UVB) to aquatic photolithotrophs and to decrease the supply of the nutrients (combined) nitrogen and phosphorus and of any non-aeolian iron. The influence of these variations on CCM expression has been examined to varying degrees as acclimation by extant organisms. Increased PAR increases CCM expression in terms of CO2 affinity, whilst increased UVB has a range of effects in the organisms examined; little relevant information is available on increased temperature. Decreased combined nitrogen supply generally increases CO2 affinity, decreased iron availability increases CO2 affinity, and decreased phosphorus supply has varying effects on the organisms examined. There are few data sets showing interactions amongst the observed changes, and even less information on genetic (adaptation) changes in response to the forcing factors. In freshwaters, changes in phytoplankton species composition may alter with environmental change with consequences for frequency of species with or without CCMs. The information available permits less predictive power as to the effect of the forcing factors on CCM expression than for their overall effects on growth. CCMs are currently not part of models as to how global environmental change has altered, and is likely to further alter, algal and aquatic plant primary productivity.

Ecological implications of microalgal and cyanobacterial CO2 concentrating mechanisms, and their regulation

The capacity of algae to express CO2 concentrating mechanisms (CCMs) is regulated by environmental factors. Some of these factors, especially photon flux, can influence the instantaneous activity of a CCM without necessarily affecting gene expression or the capacity of the cell to transport inorganic carbon. Other environmental parameters, especially those causing changes in the availability of CO2 dissolved in the surrounding medium, act at a transcriptional level. In this review, the complex interactions between environmental factors in controlling CCM activity will be discussed, as will the ecological consequences of CCMs as they relate to the growth and ecological performance of algal cells in nature. We also consider the consequences of global climate change for the performance of algae with and without CCMs.

An Anaplerotic Role for Mitochondrial Carbonic Anhydrase in Chlamydomonas reinhardtii

Previous studies of the mitochondrial carbonic anhydrase (mtCA) of Chlamydomonas reinhardtii showed that expression of the two genes encoding this enzyme activity required photosynthetically active radiation and a low CO2 concentration. These studies suggested that the mtCA was involved in the inorganic carbon-concentrating mechanism. We have now shown that the expression of the mtCA at low CO2 concentrations decreases when the external NH4+ concentration decreases, to the point of being undetectable when NH4+ supply restricts the rate of photoautotrophic growth. The expression of mtCA can also be induced at supra-atmospheric partial pressure of CO2 by increasing the NH4+ concentration in the growth medium. Conditions that favor mtCA expression usually also stimulate anaplerosis. We therefore propose that the mtCA is involved in supplying HCO3- for anaplerotic assimilation catalyzed by phosphoenolpyruvate carboxylase, which provides C skeletons for N assimilation under some circumstances.

ECOLOGICAL AND EVOLUTIONARY IMPLICATIONS OF CARBON ALLOCATION IN MARINE PHYTOPLANKTON AS A FUNCTION OF NITROGEN AVAILABILITY: A FOURIER TRANSFORM INFRARED SPECTROSCOPY APPROACH(1).

An imbalance in the cellular C:N ratio may appreciably affect C allocation in algal cells. The consequences of these rearrangements of cellular pools on cell energetics, ecological fitness, and evolutionary trajectories are little known, although they are expected to be substantial. We investigated the fate of C in 11 microalgae cultured semicontinuously at three [NO3−] and constant pCO2. We developed a new computational method for the semiquantitative use of Fourier transform infrared (FTIR) spectroscopy data for the determination of macromolecular composition. No obvious relationship was observed between the taxonomy and the allocation strategies adopted by the 11 species considered in this study. Not all species responded to a lower N availability by accumulating lipids or carbohydrates: Dunaliella parva W. Lerche and Thalassiosira pseudonana Hasle et Heimdal were homeostatic with respect to organic cell composition. A hyperbolic dependence of the lipid concentration from cell volume was observed. The level of reduction of organic constituents of green algae was parabolically related to size and was modulated in response to changes in N availability; the same was not true for the species bearing a “red” chloroplast. The above observations are discussed with respect to phytoplankton species composition and palatability for grazers, oleogenesis, and overall cell energetics.

FOURIER TRANSFORM INFRARED SPECTROSCOPY OF MICROALGAE AS A NOVEL TOOL FOR BIODIVERSITY STUDIES, SPECIES IDENTIFICATION, AND THE ASSESSMENT OF WATER QUALITY

Fourier transform infrared (FTIR) spectrometry was used to study the spectral features of 12 eukaryotic and two prokaryotic species of microalgae. The algae were cultured in liquid media containing either NO3− or NH4+ as the sole N‐source; for the NH4+ treatment, the algae were subjected to short‐term (24 h) or long‐term (1 month) incubations; for the hypersaline species, cells were also grown in the presence of 2 M NaCl. Over 500 spectra, acquired from at least three distinct cultures for each species, in each growth regime, were subjected to hierarchical cluster analysis (HCA) and were successfully separated according to their taxonomy, showing that the overall spectra were characteristic of each species and that this technique could be fruitfully employed to separate microalgal species living in a similar condition (as would be the case for a natural assemblage). In addition, in most cases, it was possible to differentiate between algae subjected to different growth treatments although belonging to the same species. We also demonstrated that it is possible to accurately identify species and determine the nutritional status of their environment of origin (e.g., N‐source), provided that suitable FTIR spectral libraries are available. This study aims to provide the basis for the development of rapid, easy, and inexpensive methods for the evaluation of biodiversity in natural phytoplankton samples and to monitor the water quality of natural environments.

CO2-CONCENTRATING MECHANISMS OF THE POTENTIALLY TOXIC DINOFLAGELLATE PROTOCERATIUM RETICULATUM (DINOPHYCEAE, GONYAULACALES)1

The low CO2 concentration in seawater poses severe restrictions on photosynthesis, especially on those species with form II RUBISCO. We found that the potentially toxic dinoflagellate Protoceratium reticulatum Clap. et J. Lachm. possesses a form II RUBISCO. To cast some light on the mechanisms this organism undergoes to cope with low CO2 availability, we compared cells grown at atmospheric (370 ppm) and high (5000 ppm) CO2 concentrations, with respect to a number of physiological parameters related to dissolved inorganic carbon (DIC) acquisition and assimilation. The photosynthetic affinity for DIC was about one order of magnitude lower in cells cultivated at high [CO2]. End‐point pH‐drift experiments suggest that P. reticulatum was not able to efficiently use HCO3− under our growth conditions. Only internal carbonic anhydrase (CA) activity was detected, and its activity decreased by about 60% in cells cultured at high [CO2]. Antibodies raised against a variety of algal CAs were used for Western blot analysis: P. reticulatum extracts only cross‐reacted with anti‐ß‐CA sera, and the amount of immunoreactive protein decreased in cells grown at high [CO2]. No pyrenoids were observed under all growth conditions. Our data indicate that P. reticulatum has an inducible carbon‐concentrating mechanism (CCM) that operates in the absence of pyrenoids and with little intracellular CO2 accumulation. Calculations on the impact of the CA activity to photosynthetic growth [CO2] suggest that it is an essential component of the CCM of P. reticulatum and is necessary to sustain the photosynthetic rates observed at ambient CO2.

Regulation of nitrate reductase in Chlamydomonas reinhardtii by the redox state of the plastoquinone pool

In the chlorophyte alga Chlamydomonas reinhardtii, expression of the nuclear gene NIA1, encoding nitrate reductase, is regulated by light, but the signal transduction mechanism is poorly understood. Using inhibitors, mutants, and physiological manipulation, we searched for signals in the photosynthetic electron transport chain that potentially regulate NIA1 expression. In the NIA1 + wild-type clone CC-1692, nitrate reductase activity is strongly down-regulated when the reduction of plastoquinone is blocked by 3-(3′4′-dichlorophenyl)-1,1′-dimethyl urea (DCMU), but unaffected or stimulated when the oxidation of plastoquinol is inhibited by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Simultaneously, although DBMIB reduced NIA1 expression by ∼30% over a 6-h period relative to the control, DCMU inhibited expression of the gene by over 80%. A cross between CC-1692 and a site-directed mutant, CC-3388 A251I, in which amino acid 251 in the PSII core protein, D1, was altered from alanine to isoleucine, thereby decreasing the binding affinity for QB, produced a cell with markedly reduced expression of NIA1. Our results indicate that expression of nitrate reductase is coupled to photosynthesis via a sensor related to the redox poise of the plastoquinone pool. When the pool is oxidized, carbon fixation is low and nitrate reductase is down-regulated; conversely, when the pool is reduced, carbon fixation is high and the gene and enzyme activity are up-regulated. These experimental observations suggest a model for the coupled light regulation of photosynthesis and nitrate assimilation.

Compositional homeostasis of the dinoflagellate Protoceratium reticulatum grown at three different pCO2

In the CO2-richer world that awaits us, the impact of elevated pCO2 on the allocation of resources in phytoplankton may have profound repercussions on the physiology of the microalgae and on the ecology of the ecosystems of which they are part. We studied the overall physiology and cell composition of the potentially toxic dinoflagellate Protoceratium reticulatum subjected to a medium-term increase of CO2. The physiological responses investigated were growth rates, cell size, photosynthetic and respiratory rates, and key enzyme activities. Cell composition was assessed by conventional analytical methods and FTIR spectroscopy. After 3 generations of incubation at current atmospheric, high and very high pCO2 (380, 1000, 5000ppm CO2), growth, photosynthesis, and dark respiration rates increased significantly, but the internal composition was only slightly affected. We propose the homeostasis of cell composition as a strategy that organisms can use to tackle environmental perturbations, especially when they are of relatively short duration.