THE INTERPLAY BETWEEN PHOTORESPIRATION AND IRON DEFICIENCY.

ABSTRACT Iron (Fe) is an essential micronutrient for plants as it takes part in major metabolic pathways such as photosynthesis and respiration and is linked to many enzymes that accomplish many other cellular functions (DNA synthesis, nitrogen fixation, hormone production). Fe deficiency reduces crop yields worldwide but particularly in plants grown on calcareous soils, which represent almost the 30% of the earth land surface. In the near future to cope with the increasing demand of food caused by a strong increase in world’s population (FAO estimates in 9 billion people by 2050), agriculture must be extended to marginal areas, many of which include calcareous soils. The most evident effect of Fe deficiency in plant leaves is a marked chlorosis caused by a decrease in chlorophyll biosynthesis, which may result in a reduction in CO2 assimilation rate. In these conditions leaves have low photo-synthetic activity but they absorb more light energy per chlorophyll mol¬ecule than required for photosynthesis, especially under high radiation. This results in a high risk for photoinhibitory and photooxidative dam¬ages in Fe-deficient leaves. The photorespiratory cycle can be considered in these circumstances as an energy dissipating cycle, operating between chloroplasts, peroxisomes, mitochondria and cytosol, which helps to protect chloroplasts from photoinhibition and plants from excessive accumulation of reactive oxygen species. We suggest that Fe deficiency leads to a strong impairment of the photosynthetic apparatus at different levels: an increase in the rate of CO2 assimilation in many biological repetition (+29%) was observed, suggesting a possible induction of photorespiratory metabolism. However, the variation was not significative and so further analysis must be required in order to reduce the variability among the repetition to get more reliable results. In addition, the reduction of CO2 assimilation can be also attributable to a reduced stomatal conductance or to a mesophyll-reduced utilization of CO2. Iron deficiency affects also amminoacid (aa) metabolism since the concentration of Ser and Gly, two aa involved in the photorespiratory metabolism, increased in leaves (+94% and +160%, respectively). Resupply of iron to Fe-deficient plants led to an increase in the concentration of some divalent cations other than Fe like Ca and Mn, whilst Na, Mg, Cu, Zn decrease as Fe sufficient condition are restored. On the other hand, as Fe deficiency proceeds during time, we observed a significant increase in Na, Mg, Zn, Mn content. This alterations suggest that Fe deficiency induces a metabolic imbalance in which other divalent cations are absorbed by unspecific transporter, due to their similar characteristics to Fe. Under our experimental conditions, ROS accumulation detected in cucumber plants grown in the absence of Fe could be attributable to an increase in the activity of enzymes involved in their formation or to a reduced detoxification. We observed a slight induction in the activity of Cu/Zn-SOD isoform whereas a reduction in Fe- and also in Mn-SOD isoforms activity was also recorded. At the same time, the concentration of H2O2 in the leaves of Fe-deficient plants was significantly higher (+40%). This overproduction could lead to an onset of oxidative stress which can lead to further cell damage at different levels also with the involvement of the photosynthetic apparatus. Fe deficiency also induces alterations in peroxisomes at different levels indicating modifications in the photorespiratory metabolism. The complete lack of Fe results in a strong inhibition of catalase activity (-35%). Nevertheless, we detect higher levels of catalase in Fe-deficient plants compared to the control condition. In Fesufficient condition the total activity of hydroxypyruvate reductase was fully attributable to the peroxisomal isoform (HPR1), while we recorded an equal distribution of the activity between the two isoforms, peroxisomal and cytosolic (HPR2) in plants grown under conditions of Fe deficiency. Moreover, the characterization of rice mutant plants defective in mitochondrial Fe importer allow us to investigate the involvement of this organelle in the photorespiratory metabolism during Fe deficiency. The partial loss of function of MIT (mit-2) affects the mitochondrial functionality by decreasing the respiratory chain activity. Furthermore, the transcriptome and the metabolome strongly change in rice mutant plants, in a different way in roots and shoot. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared to wild type plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases strongly accumulated in mit-2 plants, indicating that mit-2 mitochondria activate alternative pathways to keep the respiratory chain working. The data obtained and exposed in this doctorate thesis, in agreement with what widely previously reported in literature, allow us to state that the absence or the low Fe bioavailability during the growth of the plants results in several alterations more or less reversible at different levels of the overall metabolic plant system.