The mechanisms involved in seed dormancy alleviation by hydrogen cyanide unravel the role of reactive oxygen species as key factors of cellular signaling during germination

Abstract

The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during after-ripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release.

Figure 1.

Germination of sunflower embryos from the same seed lot at 10°C in the dark. A, Dormant embryos (dH₂O) and after-ripened nondormant embryos (ndH₂O) on water, dormant embryos treated with 0.1 mm MV (dMV), 1 mm MN (dMN), and 2.24% gaseous HCN (dHCN). B, Dormant embryos treated by HCN and DPI combined (0.1 mm dHCN + DPI), H₂O₂ (0.5 mm dH₂O₂), and AT (1 mm dAT) and nondormant embryos germinated on 0.1 mm DPI (ndDPI), 0.5 mm H₂O₂ (ndH₂O₂), and 1 mm AT (ndAT). HCN, MV, and MN treatment were carried out for 3 h before transferring the embryos to wet cotton wool at 10°C. DPI, H₂O₂, and AT were applied continuously. Data are means of three replicates ± sd.

Figure 2.

In situ localization of ROS production in the embryonic axes of sunflower embryos. A, Embryo showing axe (a) and cotyledon (c; inset frame corresponds to the view shown in B). B, Staining of embryonic axis by toluidine blue (inset frame corresponds to the views shown in confocal). C, Schematic representation of views shown in D to F. c, Cortex; p, pericycle; rc, root cap; qc, quiescent center; lrc, lateral root cap; v, vascular. D to F, Representative fluorescence images of sections of embryonic axes treated with DCFH-DA viewed by confocal laser scanning microscopy. Axes from dormant embryos imbibed for 24 h on water (D), from nondormant embryos imbibed for 24 h on water (E), and from dormant embryos treated for 3 h with cyanide and further imbibed on water (F; total treatment time was 24 h). Images correspond to maximum projections of Z planes as described in “Materials and Methods.” [See online article for color version of this figure.]

Figure 3.

Comparison of the effects of HCN and MV on transcript expression of HaNADPHox, HaPOX, HaAO1, HaAO2, HaSer/ThrPK, HaMAPK6, HaPTP, HaCaM, HaETR1, HaETR2, HaERS1, HaCTR1, and HaERF1 in embryonic axes isolated from dormant sunflower embryos after 24 h of imbibition at 10°C. Bars denote the relative transcript expression in MV-treated embryos, imbibed for 24 h at 10°C on water, which was calculated as a function of the expression found in HCN-treated embryos to which the value 1 has been assigned. Gaseous HCN and MV (1 mm) were applied during the first 3 h of imbibition. Data were produced using semiquantitative RT-PCR (HaETR1 and HaERS1) and real-time RT-PCR (all other genes) and are means of four biological replicates ± sd.

Figure 4.

A scheme showing the interactions between HCN (CN in image) and ROS in dormancy alleviation, also integrating data from Oracz et al. (2007, 2008). Positive regulators of dormancy alleviation are written in bold and negative ones in italic. ETR1, ETR2, Ethylene receptor 1 and 2, respectively; ERS1, ethylene response factor 1; CTR1, constitutive triple response 1; ERF1, ethylene response factor 1; AdoMet, S-adenosylmethionine; ACC, 1-aminocyclopropane 1-carboxylic acid; prot., protein; ox. prot., oxidized protein. [See online article for color version of this figure.]