Noninvasive Methods to Detect Reactive Oxygen Species as a Proxy of Seed Quality

Abstract

ROS homeostasis is crucial to maintain radical levels in a dynamic equilibrium within physiological ranges. Therefore, ROS quantification in seeds with different germination performance may represent a useful tool to predict the efficiency of common methods to enhance seed vigor, such as priming treatments, which are still largely empirical. In the present study, ROS levels were investigated in an experimental system composed of hydroprimed and heat-shocked seeds, thus comparing materials with improved or damaged germination potential. A preliminary phenotypic analysis of germination parameters and seedling growth allowed the selection of the best-per-forming priming protocols for species like soybean, tomato, and wheat, having relevant agroeconomic value. ROS levels were quantified by using two noninvasive assays, namely dichloro-dihydro-fluorescein diacetate (DCFH-DA) and ferrous oxidation-xylenol orange (FOX-1). qRT-PCR was used to assess the expression of genes encoding enzymes involved in ROS production (respiratory burst oxidase homolog family, RBOH) and scavenging (catalase, superoxide dismutase, and peroxidases). The correlation analyses between ROS levels and gene expression data suggest a possible use of these indicators as noninvasive approaches to evaluate seed quality. These findings are relevant given the centrality of seed quality for crop production and the potential of seed priming in sustainable agricultural practices.

Keywords: DCFH-DA; FOX-1; Glycine max; ROS; gene expression; heat-shock; seed priming; seed quality.

Figure 1

Example of the experimental system applied to Glycine max seeds. Imbibition steps are indicated in blue, dry-back is indicated in yellow, and heat-shock is indicated in orange. CTRL, non-treated control; HP2, hydropriming for 2 h; HP4, hydropriming for 4 h; HP8, hydro-priming for 8 h; HS, heat-shock.

Figure 2

Evaluation of hydropriming efficiency in Glycine max seeds. (a) Germination percentage (%). (b) Root length (cm). (c) Representative images of germinated soybean seedlings after three days of treatments. Statistical differences among treatments and control are represented with asterisks (*). p < 0.05. CTRL, non-treated control; HP2, hydropriming for 2 h; HP4, hydropriming for 4 h; HP8, hydropriming for 8 h; HS, heat-shock.

Figure 3

ROS detection in G. max seeds subjected to hydropriming and HS treatments. (a) Data collected by using the DCFH-DA fluorimetry assay and represented as relative fluorescence units (RFU). (b) Data collected from the FOX-1 assay through spectrophotometric measurements and represented as [ROOH] concentration values. Statistically significant differences (p < 0.05) are indicated by the occurrence of different letters. CTRL, non-treated control; HP2, hydropriming imbibition for 2 h; HP4, hydropriming imbibition for 4 h; HP8, hydropriming imbibition for 8 h; HP-DB, dry-back treatment following hydropriming imbibition; HS, heat-shock.

Figure 4

PCA using data gathered for the imposed treatments (CTRL, HP2, HP4, HP8, HS) for G. max. (a) Score plot grouping of samples subjected to different treatments. (b) Biplot obtained with data from germination tests (G, PV, MGT, Z, Rad) and ROS measurements (FOX-1, DCHF-DA) on the clustering of the groups subjected to the different treatments. Because the data provided consisted in triplicate values, the designation _1, _2, _3 in the plots refers to the replicate number.

Figure 5

Relative expression of genes encoding enzymes involved in ROS scavenging and production mechanisms in G. max seeds subjected to hydropriming treatments. (a) Superoxide dismutases, MnSOD and SOD1. (b) Catalases, CAT1 and CAT5. (c) Ascorbate peroxidase APX2. (d) Respiratory burst oxidase homologs, RbohE2 and RbohC2. Statistically significant differences obtained by using the Student t-test (p < 0.05) are indicated with an asterisk (*). CTRL, untreated seeds; HP4, seeds soaked for four hours in water; HP4DB, seeds soaked for four hours and subjected to the desiccation required by hydropriming protocols.

Figure 6

Pearson correlation indices calculated for G. max hydroprimed seeds taking into consideration the biochemical assays for ROS quantification (FOX-1 and DCFH-DA) and the ROS homeostasis gene (CAT1, APX2, CAT5, MnSOD, SOD1, RbohE2, RbohC2) expression levels. The blue color indicates negative correlations whereas red indicates positive correlations. Statistically significant correlations (p < 0.05) are indicated by an asterisk (*).