Responeses Of Copper/Iron-Stress Are Regulated By Nitric Oxide And Carbon Monoxide In Chlamydomonas Reinhardtii

Excess copper affects the growth and metabolism of plants and green algae. However, the physiological processes under Cu stress are largely unknown. In this study, we investigated Cu-induced nitric oxide (NO) generation and its relationship to proline synthesis in Chlamydomonas reinhardtii. The test alga accumulated a large amount of proline after exposure to relatively low Cu concentrations (2.5and5.0μM Cu2+). A concomitant increase in the intracellular NO level was observed with increasing concentrations of Cu applied. Data analysis revealed that the endogenous NO generated was positively associated with the proline level in Cu-stressed algae. The involvement of NO in Cu-induced proline accumulation was confirmed by using an NO-specific donor, sodium nitro-prusside (SNP), and an NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide]. Pre-treatment with10μM SNP increased the proline accumulation in Cu-treated cells by about1.5-fold, while this effect could be blocked by addition of10μM cPTIO. We further investigated the effect of Cu and NO on the activity and transcript amount of△1-pyrroline-5-carboxylate synthetase (P5CS, EC2.7.2.11), the key enzyme of proline biosynthesis, and observed that application of SNP was able to stimulate the P5CS activity and up-regulate the expression of P5CS in the Cu-treated algae. These results indicate that Cu-responsive proline synthesis is closely related to NO generation in C. reinhardtii, suggesting the regulatory function of NO in proline metabolism under heavy metal stress.Carbon monoxide (CO) is an endogenous gaseous molecule and regulates a variety of biological processes in animals. However, whether CO regulates nutrient stress responses in plants is largely unknown. In this paper, we described an observation that CO can regulate iron-homeostasis in iron-starved C. reinhardtii. Exogenous CO at8μM was able to prevent the iron deficient-induced chlorosis and improve chlorophyll accumulation. Expression of FTR1, FOX1, and FD was up-regulated by CO exposure in iron-deficient alge. Treatment with external CO increased iron accumulation in iron-deficient C. reinhardtii. Moreover, endogenous CO level was increased under iron-deficiency. Finally, CO exposure induced NO accumulation. However, such an action could be blocked by NO scavenger cPTIO.Generation of intracellular CO are closely linked to heme oxygenase (HO, EC1.14.99.3), which catalyzes the degradation of heme to produce CO, free iron and biliverdin. To get insight into the regulation role of HO, we constructed transgenic alge overexpressing HO-1driven by HSP70A promoter and HO-1knock out mutant named HO/OX and HO/KO respectively. Our analyses indicated that HO/OX cells could increase iron accumulation and prevent chlorosis in iron-deficient C. reinhardtii. Otherwise, the HO/KO mutant could not response to iron deficient. These results indicate that CO may play an important role in improving alga adaptation to iron deficiency or cross-talking with NO under the iron deficiency.Our data show that CO and NO may function as signal molecules to regulate the response to heavy metal stress in Chlamydomonas.