Exploring the Genotoxic Stress Response in Primed Orphan Legume Seeds Challenged with Heat Stress

Abstract

Background/objectives: The increased frequency of extreme weather events related to climate change, including the occurrence of extreme temperatures, severely affects crop yields, impairing global food security. Heat stress resulting from temperatures above 30 °C is associated with poor germination performance and stand establishment. The combination of climate-resilient crop genotypes and tailored seed priming treatments might represent a reliable strategy to overcome such drawbacks. This work explores the potential of hydropriming as a tool to mitigate the heat-stress-mediated impact on germination performance in orphan legumes.

Methods: For each tested species (Lathyrus sativus L., Pisum sativum var. arvense and Trigonella foenum-graecum L.), two accessions were investigated. Germination tests were performed at 25 °C, 30 °C, 35 °C and 40 °C to assess the heat stress tolerance threshold. Hydropriming was then applied and germination tests were performed at 40 °C to test the impact of the treatment on the seeds' ability to cope with heat stress. An alkaline comet assay and Quantitative Real Time-Polymerase Chain Reaction were performed on embryos excised from primed and control seeds.

Results: Phenotyping at the germination and seedling development stage highlighted the accession-specific beneficial impact of hydropriming under heat stress conditions. In L. sativus seeds, the alkaline comet assay revealed the dynamics of heat stress-induced DNA damage accumulation, as well as the repair patterns promoted by hydropriming. The expression patterns of genes involved in DNA repair and antioxidant response were consistently responsive to the hydropriming and heat wave conditions in L. sativus accessions.

Keywords: Lathyrus sativus L.; Pisum sativum var. arvense; Trigonella foenum-graecum L.; comet assay; heat stress; hydropriming; seed germination.

Figure 1

Overview of the experimental systems designed to assess the effects of heat waves on seeds of L. sativus accessions, Pisum sativum var. arvense accessions, and Trigonella foenum-graecum accessions. (a) Schematic representation of the experimental system designed to assess the impact of 4 h heat waves with incremental temperatures (25 °C, 30 °C, 35 °C, and 40 °C) administered after 4 h of imbibition at 24 °C. (b) Experimental system designed to assess the effects of 4 h of heat wave (40 °C) after 4 h of imbibition at 25 °C on unprimed and hydroprimed (8 h) seeds. DS, dry seed; DB, dry back; RH, rehydrated seed; UP, unprimed seeds; HP, hydroprimed seeds; NT, seeds subjected to 8 h of imbibition at 25 °C without heat wave; HW, seeds subjected to 4 h of imbibition at 25 °C followed by 4 h of heat wave at 40 °C.

Figure 2

Germination performance of the L. sativus accessions Maleme-107 and Sofades under control and heat wave conditions. (a) Germinability index (G). (b) Mean germination time (MGT). (c) Frequency of seedlings with normal and aberrant phenotypes developed from control and treated seeds. NT, non-treated with heat wave; HW, heat wave. Asterisks indicate statistically significant differences in each heat wave condition compared with the respective non-treated control, as analyzed using a heteroscedastic two-tailed Student’s t-test. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.

Figure 3

Germination performance of the P. sativum var. arvense accessions Forrimax and Guifredo under control and heat wave conditions. (a) Germinability index (G). (b) Mean germination time (MGT). (c) Frequency of seedlings with normal and aberrant phenotypes developed from control and treated seeds. NT, non-treated with heat wave; HW, heat wave. Asterisks indicate statistically significant differences in each heat wave condition compared with the respective non-treated control, as analyzed using a heteroscedastic two-tailed Student’s t-test. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.

Figure 4

Germination performance of the T. foenum-graecum accessions Rayhane and Tborsek under control and heat wave conditions. (a) Germinability index (G). (b) Mean germination time (MGT). (c) Frequency of seedlings with normal and aberrant phenotypes developed from control and treated seeds. NT, non-treated with heat wave; HW, heat wave. Asterisks indicate statistically significant differences in each heat wave condition compared with the respective non-treated control, as analyzed using a heteroscedastic two-tailed Student’s t-test. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001.

Figure 5

Germination performance of the L. sativus accessions Maleme-107 and Sofades (unprimed, hydroprimed) under control and heat wave conditions. (a) Germinability index in L. sativus Maleme-107 accession. (b) Mean germination time in L. sativus Maleme-107 accession. (c) Shoot and root length in 7-day-old seedlings in L. sativus Maleme-107 accession. (d) Germinability index in L. sativus Sofades accession. (e) Mean germination time in L. sativus Sofades accession. (f) Shoot and root length in 7-day-old seedlings in L. sativus Sofades accession. UP, unprimed seeds; HP, hydroprimed seeds; US, unstressed seeds (subjected to 8 h of imbibition at 25 °C); HW, heat wave (seeds subjected to 4 h of imbibition at 25 °C followed by 4 h of heat wave at 40 °C). G, germinability index; MGT, mean germination time. Values are indicated as average ± standard deviation. For each parameter of each accession, average values without common letters are significantly different (p-value < 0.05), as analyzed by one-way ANOVA and Duncan’s test.

Figure 6

Germination performance of the P. sativum var. arvense accessions Forrimax and Guifredo (unprimed, hydroprimed) under control and heat wave conditions. (a) Germinability index in P. sativum var. arvense Forrimax accession. (b) Mean germination time in P. sativum var. arvense Forrimax accession. (c) Shoot and root length in 7-day-old seedlings in P. sativum var. arvense Forrimax accession. (d) Germinability index in P. sativum var. arvense Guifredo accession. (e) Mean germination time in P. sativum var. arvense Guifredo accession. (f) Shoot and root length in 7-day-old seedlings in P. sativum var. arvense Guifredo accession. UP, unprimed seeds; HP, hydroprimed seeds; US, unstressed seeds (subjected to 8 h of imbibition at 25 °C); HW, heat wave (seeds subjected to 4 h of imbibition at 25 °C followed by 4 h of heat wave at 40 °C). G, germinability index; MGT, mean germination time. Values are indicated as average ± standard deviation. For each parameter of each accession, average values without common letters are significantly different (p-value < 0.05), as analyzed by one-way ANOVA and Duncan’s test.

Figure 7

Germination performance of the T. foenum-graecum accessions Rayhane and Tborsek (unprimed, hydroprimed) under control and heat wave conditions. (a) Germinability index in T. foenum-graecum Rayhane accession. (b) Mean germination time in T. foenum-graecum Rayhane accession. (c) Shoot and root length in 7-day-old seedlings in T. foenum-graecum Rayhane accession. (d) Germinability index in T. foenum-graecum Tborsek accession. (e) Mean germination time in T. foenum-graecum Tborsek accession. (f) Shoot and root length in 7-day-old seedlings in T. foenum-graecum Tborsek accession. UP, unprimed seeds; HP, hydroprimed seeds; US, unstressed seeds (subjected to 8 h of imbibition at 25 °C); HW, heat wave (seeds subjected to 4 h of imbibition at 25 °C followed by 4 h of heat wave at 40 °C); G, germinability index; MGT, mean germination time. Values are indicated as average ± standard deviation. For each parameter of each accession, average values without common letters are significantly different (p-value < 0.05), as analyzed by one-way ANOVA and Duncan’s test.

Figure 8

DNA damage (single and double strand breaks) levels in embryos excised from unprimed and hydroprimed orphan legume seeds exposed to 4 h of heat wave at 40 °C. (a) L. sativus accession Maleme-107. (b) L. sativus accession Sofades. (c) P. sativum var. arvense accession Forrimax. (d) P. sativum var. arvense accession Guifredo. (e) T. foenum-graecum accession Rayhane. (f) T. foenum-graecum accession Tborsek. UP, unprimed seeds; HP, hydroprimed seeds; US, unstressed seeds (subjected to 8 h of imbibition at 25 °C); HW, heat wave (seeds subjected to 4 h of imbibition at 25 °C followed by 4 h of heat wave at 40 °C); a.u., arbitrary units. Values are indicated as average ± standard deviation. For each accession, average values without common letters are significantly different (p-value < 0.05), as analyzed by one-way ANOVA and Duncan’s test.

Figure 9

Heat maps representing the relative expression profiles of genes involved in the seed repair response, evaluated by qRT-PCR in L. sativus accession. (a) Z-scored relative gene expression in L. sativus Maleme-107. (b) Z-scored relative gene expression in L. sativus Sofades. For each gene of each accession, expression levels without common letters are significantly different (p-value < 0.05), as analyzed by one-way ANOVA and Duncan’s test. DS, dry seed; DB, seeds subjected to hydropriming and dry-back; UP, unprimed seeds; HP, hydroprimed seeds; NT, seeds subjected to 8 h of imbibition at 25 °C without heat wave; HW, seeds subjected to 4 h of imbibition at 25 °C followed by 4 h of heat wave at 40 °C. LsSOD, superoxide dismutase. LsAPX, ascorbate peroxidase. LsMT, metallothionein. LsSPMS, spermine/spermidine synthase. LsSPDS, spermidine synthase. LsSOG1, suppressor of the gamma response 1. LsOGG1, 8-oxoguaninglycosylase/lyase. LsFPG, formamidopyrimidine-dna glycosylase. LsLig, DNA ligase. LsTOP, DNA topoisomerase. LsPCNA, proliferating cell nuclear antigen. LsTFIIS, transcription elongation factor IIS. Ls5.8S, 5.8S rRNA. Ls5.8S-IS, 5.8S rRNA-interspace. Ls, Lathyrus sativus.

Figure 10

Principal component analysis of the effects of heat stress on unprimed and hydroprimed L. sativus seeds. (a) Biplot referring to L. sativus accession Maleme-107. (b) Biplot referring to L. sativus accession Sofades. SOD, superoxide dismutase. APX, ascorbate peroxidase. MT, metallothionein. OGG1, 8-oxoguaninglycosylase/lyase. FPG, formamidopyrimidine-dna glycosylase. Lig, DNA ligase. TOP, DNA topoisomerase. TFIIS, transcription elongation factor IIS. SPMS, SPERMINE/SPERMIDINE SYNTHASE. SPDS, spermidine synthase. 5.8S, 5.8S rRNA. 5.8S-IS, 5.8S rRNA-interspace. PCNA, proliferating cell nuclear antigen. SOG1, suppressor of the γ response 1. G, germinability. MGT, mean germination time. SL, shoot length. RL, root length. SB, strand break.