Role of H2O2 in pea seed germination (2024)

Abstract

The imbibition of pea seeds with hydrogen peroxide (H₂O₂) increased the germination as well as the seedling growth, producing an invigoration of the seeds. We propose that H₂O₂ could acts as signaling molecule in the beginning of seed germination involving specific changes at proteomic, transcriptomic and hormonal levels. These findings have practical implication in the context of seed priming technologies to invigorate low vigour seeds.

Keywords: dormancy, germination, hydrogen peroxide, redox signaling, seed priming

Germination process is associated with many metabolic, cellular, and molecular events, coordinated by a complex regulatory network. The reactive oxygen species (ROS) production by germinating seeds has often been considered as a negative effect that might affect the germination process, but provided that their accumulation is tightly regulated by the balance between production and scavenging, these toxic molecules now appear as being beneficial for germination.¹

Strategies for improving the growth and development of crop species have been investigated for many years. Seed priming is a pre-sowing strategy to influence seed germination and seedling development by modulating pre-germination metabolic activity prior to emergence of the radicle and generally enhances germination rate and plant performance.^2,3 From a biochemical and molecular point of view, studying germination is difficult because a population of seeds does not complete the process synchronously.⁴ Seed priming has been found as technology to enhance rapid and uniform emergence, and to achieve high vigour and better yields. This process generally causes faster germination and faster field emergence, which has practical agronomic implications, notably under adverse germination conditions.⁵

Effect of ROS-Related Compound in Seed Germination and Seedling Growth

We have tested the priming effect of several compounds on pea seeds. The assayed compound can be divided in three major groups: directly involved in the antioxidative metabolism [hydrogen peroxide (H₂O₂), reduced and oxidized glutathione (GSH, GSSG) and ascorbic acid (ASC)]; compounds related with the amino acid cysteine [N-acetylcysteine (NAC), thioproline (TP) and L-2-oxo-4-thiazolidine-carboxylic acid (OTC)]; and compounds that trigger the systemic acquired resistance in plants [salicylic acid (SA) and its functional analog benzothiadiazole (BTH)].

The tripeptide thiol glutathione (GSH) is at the hub of the complex antioxidant networks of plant and animal cells, where it participates in the cellular redox signaling networks that influence growth, development and defense.^6,7 It has long been recognized that ASC also exert a strong influence on plant growth and development.⁸ The GSH synthesis is regulated by cysteine availability, thus compounds increasing its contents could produce an increase of GSH levels. In this sense, OTC is an artificial cysteine precursor whereas TP is a proline analog that is converted by proline oxidase to N-formyl-l-cysteine which is presumably hydrolized to cysteine.⁹

To carry out this work, pea seeds were imbibed for 24 h in dH₂O or in the compounds described above at different concentrations. Seeds were then washed twice with dH₂O and placed in Petri dishes with two layers of filter paper moistened with dH₂O. Seeds were incubated at 25°C for 48 h in darkness, in a Cooled Incubator (MIR-153 Sanyo).

Figure1 shows that, except H₂O₂, none of the assayed compounds had a positive effect on seed germination or seedling growth (measured as fresh weight and length). Exogenous H₂O₂ showed a priming effect in the germination of pea seeds in a concentration dependent-manner obtaining more vigorous seedlings, being 20mM H₂O₂ the concentration that produced the best response in terms of growth (Fig.1). The increase in seedling growth by 20mM H₂O₂ was also evident 24 h after imbibition (Fig.2). The priming effect of H₂O₂ was also noticeable at shorter times of imbibition (Table 1). After 12h of imbibition about 15% of seeds had germinated; however, no germination occurred at this time in seeds imbibed in water. At 24h of imbibition, this percentage had reached nearly 75%, whereas control seed germination remained at low level (Table 1). In preliminary experiments, we noticed that H₂O₂ concentrations higher than 20 mM (40 to 100mM) also stimulated the germination rate after 24h imbibition. However, at short-term of post-imbibition (6h) we observed that these H₂O₂ levels induced a pronounced curvature as well as an abnormal growth of the radicle at 24h and 48h post-imbibition (Fig.2). In addition, H₂O₂ concentrations above 100mM reduced the pea seeds germination rate (data not shown).

Interestingly, we observed a differential response depending on when H₂O₂ was supplied. When 5mM H₂O₂ was added during the incubation in plates, after imbibition in dH₂O, the percentage germination was similar to that of control seeds. In contrast, the presence of 10or20mM H₂O₂ in Petri dishes produced a negative effect on germination (Fig.3).

H₂O₂ Signaling during Seed Germination

Numerous recent works show now that ROS would play a key signaling role in the achievement of major events of seed life, such as germination or dormancy release. In fact, hydrogen peroxide, nitric oxide, hydroxyl radicals and superoxide radicals have been shown to accumulate during seed germination in various species.¹ Many works have reported that exogenous application of H₂O₂ can improve seed germination in many plant species.^10-12 The interplay between ROS and hormone signaling pathways lead to changes in gene expression or in cellular redox status that would play a role in the perception of environmental factors by seeds during their germination.¹ Recently we have shown that H₂O₂ coordinates the beginning of pea seed germination, acting as a priming factor that involves specific changes at proteome, transcriptome and hormonal levels, resulting in an acceleration of the germination process most probably due to invigoration of the seeds.¹³ H₂O₂ would induce a MAPK-dependent decrease in abcisic acid (ABA) contents in the seed as well as the carbonylation of seed storage proteins,¹³ favoring their mobilization, and some glycolytic enzymes that could stimulate the phosphate pentose pathway (oxPPP).¹⁴ The oxPPP activation could provide NADPH for the thioredoxin system, involved in seed germination and seedling development.¹⁵ Alternatively, H₂O₂ could act, directly or indirectly impairing the ABA transport from the cotyledon to the embryo inducing a decrease in ABA, stimulating the germination process.¹³ Finally, the decrease in ABA could induce a MAPK-mediated decrease in the ethylene precursor 1-aminocyclopropane carboxylic acid, favoring epicotyl and radicle emergence by H₂O₂ treatment.¹³

As mentioned above, the H₂O₂ treatment also stimulates the early growth of pea seedlings (Table1). Previously, we have described that the H₂O₂-induced increase in pea seedling growth was correlated with the induction of proteins related to plant growth, cellular signaling and cell cycle control (14–3-3 protein, profilin, proteasome, translationally-controlled tumor protein), as well as with a substantial decrease in the levels of the hormones ABA and zeatin-riboside (ZR).¹⁶ Moreover, a decrease of a polypeptide with hom*ology to an ABA-responsive protein was observed, suggesting an interplay between the redox state and plant hormones, orchestrated by H₂O₂, in the induction of proteins related to plant signaling and development during the early growth of pea seedlings.¹⁶ The molecular mechanisms implicated in this differential response remain unclear. They are of considerable interest, notably in the context of improving crop yields by invigoration seed treatments both in commercial applications² and in developing countries.¹⁷

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

This work was supported by Fundacio´n Se´neca—Agencia de Ciencia y Tecnologi´a de la Regio´n de Murcia (projects 05571/PI/07 and 11883/PI/09). G.B.-E. thanks CSIC for his JAE research fellowship. P.D.-V. thanks CSIC for his JAEdoc research contract.

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