Case Study

Metabolic and Gene Expression Hallmarks of Seed Germination

Metabolon helps identify differentially accumulated metabolites that could be utilized as potential metabolic signatures of seed germination under adverse conditions.

Metabolon’s nontargeted global metabolomics profiling revealed stress-induced changes in seed metabolism, which can provide novel potential metabolic hallmarks of germination.

Metabolon’s nontargeted global metabolomics profiling revealed stress-induced changes in seed metabolism, which can provide novel potential metabolic hallmarks of germination.

Modern Farming Agricultural Science Seed Germination

The quality of seed crops is determined by seed vigor—rapid, uniform, and robust germination and growth across diverse environmental conditions. Not surprisingly, improving seed vigor is important because crop yield relies on enhancing the critical and yield-defining stage of crop growth. High-quality seeds are essential for successful crop production, especially in challenging environments.

While the molecular mechanisms that determine the vigor of seed germination are poorly understood, studies support the idea that damaged cell structures and macromolecules contribute to deterioration of seed quality.1 The amount of DNA damage accumulated in the embryo genome and the seed’s capacity to repair this damage are major determinants of seed vigor. The DNA damage response in particular, is now recognized as an important regulator of germination. Filling the gaps in our understanding of the molecular basis of seed vigor will help advance seed technology, which will be critical to meeting the ever-growing demands to produce crops in challenging environments. One model known as Medicago truncatula has been used to study the unique biological mechanisms that legumes use to respond to stress2 including the plants’ DNA damage response (DDR).3-6

Histone acetyltransferases and histone deacetylases (HDAC) establish and remove histone acetylation, respectively.7 In plants, histone acetylation/deacetylation plays a role in many physiologic processes, including rapid responses to internal or external signals8,9 or the switch from vegetative to reproductive growth.10 HDACs are involved in aspects of DNA repair. Thus, HDAC inhibitors, which cause histone hyperacetylation and transcription activation, are used to explore molecular pathways of abiotic stress response in plants.11,12 In animal cells, HDAC inhibitors have helped us understand the DDR in response to genotoxic stress.13,14 These aspects of stress response have been characterized in animal cells, but not in plant cells during seed germination.

The Challenge: Molecular Basis Not Well Understood

The DDR in seeds includes a coordinated network of reactions to help minimize the consequences of damage to the genome of the cells. Responses may include cell cycle checkpoints, DNA repair factors, and programmed cell death (or apoptosis). DDR is triggered during early seed imbibition to ensure the integrity of the genome is maintained. This ultimately contributes to seed quality. However, metabolomic studies, especially those investigating the effects of HDAC inhibitors on seed response, are lacking, and investigators need these types of studies to identify early markers of seed vigor and further characterize abiotic stressors, as described below.

Metabolon Insight: Molecular Phenotyping Leveraged to Assess Seed Response to Stress

A study conducted by Pagano et al.15 investigated novel signatures of the seed response in M. truncatula, using the HDAC inhibitor sodium butyrate (NaB) as a stress agent. The researchers leveraged multiple molecular phenotyping methods that included: electron paramagnetic resonance (to detect free radical species in seeds), the comet assay (to assess DNA damage), and qRT-PCR (gene expression), and untargeted metabolomics analysis.15

The Solution: Metabolomics Reveals Stress-induced Changes in Seed Metabolism

NaB-treated and untreated seeds were collected at 2 and 8 hr. of imbibition and at the radical protrusion stage and then analyzed. NaB treatment impaired seed germination and seedling growth but did not impact free radical steady-state levels in germinating seeds. The antioxidant response, reflected by upregulation of several antioxidant (AOX) genes, was significantly higher in NaB-treated seeds. It is unclear whether the upregulation depends on reactive oxygen species (ROS). NaB treatment damaged DNA, as evidenced by strands breakage and expression of base excision repair (BER) genes, but it is unknown whether this is a consequence of oxidative stress or histone hyperacetylation. The Global Discovery Panel revealed stress-induced changes in seed metabolism. NaB treatment impacted nucleotide, amino acid, lipid, and carbohydrate metabolism at the radical protrusion stage, with the most significant changes noted for nucleotide and amino acid metabolism. Although samples clustered together, using principal component analysis based on imbibition time points, a heatmap with hierarchical clustering showed trends between water and NaB treated samples. Groupings based on treatment type were evident when looking at significantly different metabolites on the heatmap, with amino acids being one class of metabolite that showed strong changes. The uracil metabolite (3-ureidopropionate) was one of the metabolites that contributed significantly to the separation between control and NaB-treated samples.

The Solution: Differentially Accumulated Metabolites Potential Hallmarks of Seed Germination

Differentially accumulated metabolites associated with polyamine biosynthesis and uracil degradation could be utilized as potential metabolic signatures of seed germination under adverse conditions because of their documented roles in cellular stress response.

The Outcome: Metabolomics Provides Possible Metabolic Signatures of Seed Germination Under Adverse Conditions

These results helped to advance our understanding of the molecular basis of the stress-induced DDR in seed during germination. Further studies are needed to validate the differentially accumulated metabolites as biomarkers and to develop new seed invigoration treatments.

References

1. Waterworth WM, Bray CM, West CE. Seeds and the Art of Genome Maintenance. Front Plant Sci. 2019;10:706. Published 2019 May 31. doi:10.3389/fpls.2019.00706

2. Araujo SS, Beebe S, Crespi M, et al. Abiotic stress responses in legumes: strategies used to cope with environmental challenges. Critical Reviews in Plant Sciences. 2015;34(1-3):237-280. doi:10.1080/07352689.2014.898450

3. Confalonieri M, Faè M, Balestrazzi A, et al. Enhanced osmotic stress tolerance in Medicago truncatula plants overexpressing the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2). Plant Cell, Tissue and Organ Culture (PCTOC). 2014/02/01 2014;116(2):187-203. doi:10.1007/s11240-013-0395-y

4. Donà M, Balestrazzi A, Mondoni A, et al. DNA profiling, telomere analysis and antioxidant properties as tools for monitoring ex situ seed longevity. Ann Bot. 2013;111(5):987-998. doi:10.1093/aob/mct058

5. Faè M, Balestrazzi A, Confalonieri M, et al. Copper-mediated genotoxic stress is attenuated by the overexpression of the DNA repair gene MtTdp2α (tyrosyl-DNA phosphodiesterase 2) in Medicago truncatula plants. Plant Cell Rep. 2014;33(7):1071-1080. doi:10.1007/s00299-014-1595-6

6. Sabatini ME, Donà M, Leonetti P, et al. Depletion of tyrosyl-DNA phosphodiesterase 1α (MtTdp1α) affects transposon expression in Medicago truncatula. J Integr Plant Biol. 2016;58(7):618-622. doi:10.1111/jipb.12457

7. Wang Z, Cao H, Chen F, Liu Y. The roles of histone acetylation in seed performance and plant development. Plant Physiol Biochem. 2014;84:125-133. doi:10.1016/j.plaphy.2014.09.010

8. Benhamed M, Bertrand C, Servet C, Zhou DX. Arabidopsis GCN5, HD1, and TAF1/HAF2 interact to regulate histone acetylation required for light-responsive gene expression. Plant Cell. 2006;18(11):2893-2903. doi:10.1105/tpc.106.043489

9. Hollender C, Liu Z. Histone deacetylase genes in Arabidopsis development. J Integr Plant Biol. 2008;50(7):875-885. doi:10.1111/j.1744-7909.2008.00704.x

10. He Y, Michaels SD, Amasino RM. Regulation of flowering time by histone acetylation in Arabidopsis. Science. 2003;302(5651):1751-1754. doi:10.1126/science.1091109

11. Patanun O, Ueda M, Itouga M, et al. The Histone Deacetylase Inhibitor Suberoylanilide Hydroxamic Acid Alleviates Salinity Stress in Cassava. Front Plant Sci. 2017;7:2039. Published 2017 Jan 9. doi:10.3389/fpls.2016.02039

12. Wang P, Zhao L, Hou H, et al. Epigenetic Changes are Associated with Programmed Cell Death Induced by Heat Stress in Seedling Leaves of Zea mays. Plant Cell Physiol. 2015;56(5):965-976. doi:10.1093/pcp/pcv023

13. Roos WP, Krumm A. The multifaceted influence of histone deacetylases on DNA damage signalling and DNA repair. Nucleic Acids Res. 2016;44(21):10017-10030. doi:10.1093/nar/gkw922

14. Chen CC, Huang JS, Wang TH, et al. Dihydrocoumarin, an HDAC Inhibitor, Increases DNA Damage Sensitivity by Inhibiting Rad52. Int J Mol Sci. 2017;18(12):2655. Published 2017 Dec 7. doi:10.3390/ijms18122655 

15. Pagano A, de Sousa Araújo S, Macovei A, Dondi D, Lazzaroni S, Balestrazzi A. Metabolic and gene expression hallmarks of seed germination uncovered by sodium butyrate in Medicago truncatula. Plant Cell Environ. 2019;42(1):259-269. doi:10.1111/pce.13342

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