DOI: 10.1007/s11099-016-0197-7 PHOTOSYNTHETICA 54 (3): 468-474, 2016 468 Influence of exogenous 5-aminolevulinic acid on chlorophyll synthesis and related gene expression in oilseed rape de-etiolated cotyledons under water-deficit stress D. LIU * , D.D. KONG ** , X.K. FU * , B. ALI ** , L. XU *** , and W.J. ZHOU **,+ Key Laboratory for Tobacco Gene Resources, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, China * College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China ** College of Life Sciences and Zhejiang Key Laboratory of Plant Secondary Metabolism and Regulation, Zhejiang Sci-Tech University, Hangzhou 310018, China *** Abstract 5-aminolevulinic acid (ALA) is an essential precursor for the biosynthesis of tetrapyrrols such as heme and chlorophyll (Chl). Previous studies have focused mainly on promotive effects of exogenous ALA on plant growth, while regulatory mechanisms affecting Chl biosynthesis have been only partially discussed. In the present study, the ameliorative role of exogenous ALA was investigated on Chl and endogenous ALA biosynthesis in six-day-old etiolated oilseed rape (Brassica napus L.) cotyledons during the de-etiolation stage. We showed that exogenously applied ALA of a low dosage enhanced Chl and ALA accumulation in cotyledons, while 600 μM ALA treatment inhibited the accumulation of Chl and ALA severely. However, the gene expression levels of glutamyl-tRNA reductase (HEMA) and glutamate-1-semialdehyde aminotransferase (GSA) were not affected under either low or high ALA concentrations. Furthermore, water deficit induced by polyethylene glycol 6000 (PEG) suppressed the Chl and ALA accumulation in cotyledons, while the inhibition was partially alleviated in the cotyledons pretreated with ALA. The decrease in Chl biosynthesis induced by PEG stress was assumed to be related to downregulation of HEMA and Mg-chelatase ChlH (ChlH), and upregulation of ferrochelatase (FC) genes. Moreover, exogenously applied ALA did not show any effect on the expression of Chl synthesis-related genes under the PEG treatment. These results showed a difference in suppressing ALA synthesis due to the high concentration of ALA and PEG. Exogenously applied ALA did not affect the expression of HEMA and GSA, thus exogenous ALA regulated Chl synthesis not via the regulation of transcriptional level in ALA biosynthesis. However, the inhibition in Chl and endogenous ALA accumulation by the PEG treatment may be attributed to downregulation of HEMA and ChlH, and upregulation of FC. Additional key words: protochlorophyllide; rapeseed; transcript; uroporphyrinogen decarboxylase; water stress. Introduction 5-aminolevulinic acid (ALA) is an essential precursor in tetrapyrrole biosynthesis. Treatment of etiolated angio- sperm cotyledons with exogenous ALA caused accumula- tion of protochlorophyllide (Pchlide) (Granick 1959). Enzymes required for Pchlide synthesis are already active and present in nonlimiting amounts in etiolated plant tissues and only the amount and activity of enzymes involved in ALA synthesis limit the synthesis rate (Granick 1959, Papenbrock and Grimm 2001). The reduction of Pchlide to chlorophyllide is catalyzed by the light-dependent NADPH:Pchlide oxidoreductase (POR) that requires light for its catalysis activity (Schoefs and Franck 2003, Masuda ——— Received 9 May 2015, accepted 18 December 2015, published as online-first 18 January 2016. + Corresponding author; e-mail: wjzhou@zju.edu.cn Abbreviations: ALA – 5-aminolevulinic acid; Chl – chlorophyll; ChlH – gene encoding Mg-chelatase ChlH subunit; FC – gene encoding ferrochelatase; FLU – fluorescent; FM – fresh mass; GluTR – glutamyl-tRNA reductase; GSA – gene encoding glutamate-1- semialdehyde aminotransferase; HEMA – gene encoding glutamyl-tRNA reductase; Lhcb – LHC protein of PSII type III chlorophyll a/b-binding protein; MgCh – Mg-chelatase; Pchlide – protochlorophyllide; PEG – polyethylene glycol; POR – protochlorophyllide oxidoreductase; qRT-PCR – quantitative real-time PCR; URO – gene encoding uroporphyrinogen decarboxylase. Acknowledgments: This work was supported by the Special Fund for Agro-scientific Research in the Public Interest (201303022), Jiangsu Collaborative Innovation Center for Modern Crop Production, the National Natural Science Foundation of China (31170405, 31301678, 31570434), and the Science and Technology Department of Zhejiang Province (2012C12902-1, 2011R50026-5).