Nitric Oxide Protect Maize Seedlings Against Iron Deficiency Stress

It is a severe problem in alkaline soil that iron deficiency leads to the receded quality and the reduced production of plant. It is related to health of human being and the safety of the food in the world. The previous researches were focused on the physiological and biochemical changes on the plant due to iron deficiency. But it was not studied well enough to understand the exact molecular mechanism of injury due to iron deficiency. Even though it is the hotspot on the study of nitric oxide in plant kingdom, there is less reports on the mechanism of NO on the reverse of iron-deficiency induced chlorosis.Physiological and biochemical changes of monocotyledonous plant maize (Zea mays L.) were studied and the protective effects of nitric oxide (NO) against iron deficiency induced injury were analyzed simutaniously. The plants were grown in monofactor controlled solutions using solution cultural method and the full expanded fourth leaves were used in all of the experiments. The biochemical and the molecular mechanisms of iron induced injury and the protective mechanisms of NO against iron deficiency induced injury were studied. These results have important theoretical and practical values in the research of iron-in-plant and in the research of protective mechanisms of NO on plants grown under iron deficiency.Because iron is the pivotal element in chlorophyll biosynthesis, iron deficiency leads to decreased content of chlorophylls in leaves, decreased proteins contents in chloroplast, and the reduced growth of plants. It was evident in our experiment that the plant was chlorosis due to iron deficiency. Transmission electron microscope (TEM) analysis showed that the thylakoid was not developed well in iron-deficient maize leaves and the grana were not well stacked. Iron deficiency lead to restrained development of stromal thylakoid and increased numbers of the osmiophilic globule (Og). Some broken thylakoids were found in severe iron-deficient leaves. However the leaves treated with NO had normal level of chlorophyll contents and retained normal chloroplast. The stromal thylakoid and the grana in chloroplast were developed well enough, and the osmiophilic globule reduced to small particles observed from TEM pictures. These results indicated that NO reversed the iron induced injury and protected plants against iron deficiency stress.Chlorophyll fluorescence analysis showed that the active reaction centers of PSII were reduced, the connection between the PSII reaction center and the antenna was impaired. The greatly reduced of maximal photochemical efficiency and the actual photochemical efficiency of PSII, the decreased photochemical quenching and the increased nonphotochemical quenching of chlorophyll fluorescence sugguested that the PSII itself was damaged and the electron transport chain was damaged, too. Blue native gel and denaturing gel SDS-PAGE analysis showed the greatly reduced protein in chloroplast thylakoid and the loss of integrality of some chlorophyll protein complexes. From these complexes the iron containing complexes such as PSII complex and Cytb₆/f complex of electron transport chain was damaged severely. No PSII supercomplex was formed in the plant grown in iron depletion solution. After treatment with NO, all of the above injuries of plant due to iron deficiency were reverted, such as the increase of active reaction center of PSII, the reconnection of PSII with antenna, the increased electron transport rate, the increased photochemical fluorescence quenching and the reduced non-photochemical fluorescence quenching. Protein electrophoresis revealed that the damaged protein was repaired due to NO treatment. Our results revealed the protective effect of NO against iron deficientcy induced damage.Although the reduced radiation obtained by chlorosis leaves due to less chlorophyll under iron deficiency, overexcitation occured in leaves because the hindered electron transport chain. Thus reactive oxygen species (ROS) such as superoxide radical (O₂^.-) and hydrogen peroxide (H₂O₂) were produced in iron-inefficient leaves, which probably the reasons of damage. ROS react with membrane lipid to induce lipid peroxidation and with membrane protein to induce injury of some important proteins, which ultimately destroy the plant. Because iron is cofactor of ascorbate-dependent peroxidase (APX), peroxidase (POD) and catalase (CAT), iron deficiency reduced the activities of these three enzymes. However, the activity of superoxide dismutase (SOD) was increased under iron deficiency. The enhanced content of ascorbate acid (ASC) and the increased activity of glutathione reductase (GR) indicated that the capacity of ascorbate-gluthione (ASC-GSH) cycle was enhanced. Based on these results we can deduce that SOD, ASC-GSH cycle and some small molecules such as ASC was the main role in scavenging ROS in iron deficient maize leaves. Treatment with NO reduced O₂^,- and H₂O₂, and improved the activity of APX, POD and CAT. We postulated that NO scavenge ROS directly and/or by improving the activity of APX, POD and CAT to protect plant against oxidative stress.