Effects of Exogenous 2,4-Dichlorophenoxyacetic Acid, Glutathione, and L-Buthionine Sulfoximine on Intracellular Redox State and Hormone Levels and Their Links with Proliferation in Picea pungens Somatic Embryogenesis

Cellular redox state and hormone levels have important regulatory roles in the process of somatic embryogenesis (SE), but experimental data on the topic are limited in conifer SE. This study investigated changes in redox pairs (reduced glutathione (GSH) vs oxidized glutathione (GSSG) and ascorbic acid (ASA) vs dehydroascorbate (DHA)), activities of enzymes (glutathione reductase (GR), DHA reductase (DHAR), and ascorbate peroxidase (APX)), and levels of endogenous hormones (indole-3-acetic acid (IAA), zeatin-riboside (ZR), gibberellin (GA), and abscisic acid (ABA)) following exogenous supplements of 2,4-dichlorophenoxyacetic acid (2,4-D), GSH, and L-buthionine sulfoximine (BSO) to the proliferation medium of Picea pungens SE and linked the changes with proliferation efficiencies. While 2,4-D promoted proliferation and maturation in a concentration-dependent manner, the joint application of 2,4-D (2 mg/L) and GSH (0.5 mmol/L) was the most effective. Such beneficial roles were not observed when GSH alone was applied and exogenous BSO (1 mmol/L) prohibited proliferation. Supplements of 2,4-D and GSH, individually or jointly, enhanced the GR activity, decreased the DHAR activity and increased amounts of redox compounds, yet more in GSH and ASA than in GSSG and DHA, creating reduced environments. Exogenous BSO had reversal effects, creating an oxidized environment, and so was the joint use of 2,4-D and BSO, although at a less extent. 2,4-D and GSH reduced while BSO increased IAA content. The alternations in redox state as well as in IAA content from exogenous 2,4-D or BSO supplements coincided with the corresponding changes in proliferation. The notable finding was that exogenous GSH, regardless its substantial roles in reducing environments or IAA content, did not improve proliferation. Thus, the role of GSH on proliferation varied, depending on 2,4-D existing, and could not be fully accounted for by dynamic changes in redox state or hormone level. Alternations in APX and hormones ABA, ZR, GA, and IAA/ABA ratio were also found not highly involved in altering proliferation.