Peroxidase Activity as Reliable Indicator for Evaluating Storability of Japonica Rice Seeds

doi:10.1016/j.rsci.2026.05.011

Abstract

Abstract:

Seed storability is a critical factor influencing the long-term viability of seeds in genebanks. However, evaluating seed storability remains challenging, as current methods such as viability loss curves are time-consuming and require substantial seed resources. This study investigated the role of phenotypic and physiological factors in seed storability using 56 japonica rice seed samples with initial germination rates exceeding 90%. Our analysis revealed that phenotypic traits, such as 1000-grain weight, grain width, and length-to-width ratio, did not significantly correlate with seed storability, highlighting the complexity of the relationship between phenotype and storability. Dynamic changes in antioxidant enzyme activities [superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), peroxidase (POD), and glutathione reductase (GR)] were analyzed, and a significant positive correlation was observed between POD activity and seed storability. POD activity remained relatively high in storage-tolerant seeds, whereas it decreased more sharply in storage-sensitive seeds. Gene expression analysis of 11 highly expressed POD genes in seeds generally reflected the dynamic change in POD activity. A linear regression model constructed using POD activity and initial germination rate showed that POD activity could reliably assess seed storability, with an R² value of 0.919 for storage-tolerant seeds. Cluster analysis of 28 seed samples validated these findings, establishing a POD activity threshold of 12 U/μg protein in unaged seeds imbibed for 48 h as a key indicator for distinguishing storage-tolerant seeds. This study provides valuable insights into the physiological and genetic mechanisms underlying seed storability and offers a promising approach for rapidly assessing japonica rice seed storability in genebanks.

Key words: seed storability, japonica rice, peroxidase activity, antioxidant system, seed viability loss

Zhao Yunxia, Gao Yanfeng, Gao Cheng, Li Yang, Hu Mingyu, Li Ming, Zhang Jinmei, Xin Xia, Yin Guangkun. Peroxidase Activity as Reliable Indicator for Evaluating Storability of Japonica Rice Seeds[J]. Rice Science, 2026, 33(5): 701-714.

Figures/Tables 10

Fig. 1. Seed viability loss curves and storability classification of 56 japonica rice seeds. A, Seed viability loss curves of 56 japonica rice seeds. Changes in seed germination rate of 56 japonica rice seeds subjected to accelerated aging at 40 ºC and 75% relative humidity for 30 d. B, Storability classification of 56 japonica rice seeds. The 56 japonica rice seeds were classified into three categories: blue represents storage-tolerant seeds, green represents intermediate seeds, and red represents storage-sensitive seeds. The numbers outside the circle are the rice germplasm accession numbers (e.g., 1, 2, 3), and the radial scale inside the circle (0, 50, 100, 150, 200, 250, and 300) denotes the magnitude of the corresponding trait value.

Table 1. Variation in phenotypic traits of 56 japonica rice seeds.

Grain trait	Maximum	Minimum	Mean	Range	Standard deviation
1000-grain weight (g)	31.64	20.35	24.80	11.29	2.63
Length (mm)	9.16	6.71	7.69	2.45	0.52
Width (mm)	4.00	2.89	3.47	1.11	0.18
Length-to-width ratio	2.82	1.86	2.23	0.96	0.20
Area (mm²)	21.90	15.36	17.92	6.54	1.49
Perimeter (mm)	24.36	17.62	20.05	6.74	1.32
Roundness	0.66	0.45	0.58	0.21	0.05

Fig. 2. Correlation between grain phenotypic traits and germination rate after japonica rice seeds accelerated aging for 30 d. TGW, 1000-grain weight; CN, Critical node. 0‒30 d refer to aging times. Correlation analysis method is ellipse. Data are mean ± SD (n = 3) according to Student’s t-test (*, P ˂ 0.05).

Fig. 3. Comparison of activities of catalase (CAT, A), superoxide dismutase (SOD, B), ascorbate peroxidase (APX, C), glutathione reductase (GR, D), and peroxidase (POD, E) in non-imbibed seeds of five storage-tolerant and five storage-sensitive japonica rice both non-aged (control) and aged to critical node of viability. Data are mean ± SD (n = 3). All analyses were conducted separately for the control and critical node treatments. Bars marked with different uppercase letters differ significantly among accessions under the control treatment; bars marked with different lowercase letters differ significantly among accessions under the critical-node treatment. Bars sharing the same letter (same case) show no significant difference (P < 0.05, Duncan’s multiple-range test).

Fig. 4. Comparison of activities of catalase (CAT, A), superoxide dismutase (SOD, B), ascorbate peroxidase (APX, C), glutathione reductase (GR, D), and peroxidase (POD, E) in 48-h-imbibed seeds of five storage-tolerant and five storage-sensitive japonica rice both non-aged (control) and aged to critical node of viability. Data are mean ± SD (n = 3). All analyses were conducted separately for the control and critical node treatments. Bars marked with different uppercase letters differ significantly among accessions under the control treatment; bars marked with different lowercase letters differ significantly among accessions under the critical-node treatment. Bars sharing the same letter (same case) show no significant difference (P < 0.05, Duncan’s multiple-range test).

Fig. 5. Correlation analysis between antioxidant enzyme activities [catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), peroxidase (POD)] and germination rate in japonica rice seeds after accelerated aging for 30 d. A, Seeds imbibed for 48 h. B, Non-imbibed seeds. CN, Critical node. 0‒30 d refer to aging times. Correlation analysis method is ellipse. Data are mean ± SD (n = 3) according to Student’s t-test (*, P ˂ 0.05).

Fig. 6. Relative expression levels of 11 POD genes in 5 storage-tolerant and 4 storage-sensitive japonica rice seeds at the critical node of viability compared with non-aged seeds after imbibition for 48 h. Relative expression = -2-[ΔCN(gene ‒ ubq5) ‒ ΔCK(gene ‒ ubq5)]. Samples were collected from seed embryos in non-aged seeds and seeds aged to the critical node of viability. qRT-PCR was performed with Ubq5 as the internal reference gene. Data are mean ± SD (n = 3). Lowercase letters above bars indicate significant differences according to Duncan’s multiple range test at P < 0.05.

Fig. 7. Comparison of peroxidase activity of 28 japonica rice accessions under non-aging treatment between non-imbibed and 48-h-imbibed seeds. Data are mean ± SD (n = 3). According to Duncan’s multiple range test at P < 0.05, different lowercase letters above bars indicate statistically significant differences among different germplasms.

Table 2. Regression equation for peroxidase (POD) activity-based storability evaluation in japonica rice seeds.

Variable	R²	Equation
POD activity at 0 and 48 h after imbibition, germination rate	0.423	Y = 0.345X₁ ‒ 0.058X₂ + 0.422X₃
POD activity at 0 h after imbibition, germination rate	0.340	Y = 0.541X₁ + 0.153X₂
POD activity at 48 h after imbibition, germination rate	0.421	Y = 0.358X₂ + 0.382X₃
POD activity of storage-tolerant seeds at 48 h after imbibition, germination rate	0.919	Y = 0.899X₂ ‒ 0.326X₃
POD activity of storage-intermediate seeds at 48 h after imbibition, germination rate	0.103	Y = 0.247X₂ + 0.164X₃
POD activity of storage-sensitive seeds at 48 h after imbibition, germination rate	0.228	Y = -0.471X₂ + 0.008X₃

Fig. 8. Hierarchical clustering based on initial germination rate and peroxidase activity after 48 h of imbibition. The clustering analysis divides the samples into three categories: red represents storage-tolerant seeds, green represents intermediate seeds, and blue represents storage-sensitive seeds. The scale values represent how different the samples are from each other (the distance), the bigger the number, the greater the difference between the two samples in terms of the ‘storability’ metric; the smaller the number, the more similar they are.

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