Plant and Cell Physiology Advance Access originally published online on February 5, 2008
Plant and Cell Physiology 2008 49(3):411-419; doi:10.1093/pcp/pcn017
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Copper-Induced Proline Synthesis is Associated with Nitric Oxide Generation in Chlamydomonas reinhardtii
1Department of Biochemistry and Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing 210095, PR China
2Key Lab of Marine Biology in Jiang Su, College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, PR China
*Corresponding author: E-mail, zmyang{at}njau.edu.cn
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Abstract |
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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 (2.5 and 5.0 µM 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 nitroprusside (SNP), and an NO scavenger cPTIO [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide]. Pre-treatment with 10 µM SNP increased the proline accumulation in Cu-treated cells by about 1.5-fold, while this effect could be blocked by addition of 10 µM cPTIO. We further investigated the effect of Cu and NO on the activity and transcript amount of 
1-pyrroline-5-carboxylate synthetase (P5CS, EC 2.7.2.11
[EC]
), 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.
Keywords: Chlamydomonas reinhardtii - Copper - Nitric oxide - Proline
Abbreviations:
cPTIO, 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide; DAF-2DA, 4,5-diaminofluorescein diacetate; NO, nitric oxide; NOS, nitric oxide synthase; P5CS, 1-pyrroline-5-carboxylate synthetase; ROS, reactive oxygen species; RT–PCR, reverse transcription–PCR; SNP, sodium nitroprusside.
3These authors contributed equally to this work.
(Received November 11, 2007; Accepted January 28, 2008)
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