Alternating temperature breaks dormancy in leafy spurge seeds and impacts signaling networks associated with HY5

Non-after-ripened seeds of the herbaceous perennial weed leafy spurge do not germinate when imbibed at a constant temperature (C), but transfer to an alternating temperature (A) induced germination. Changes in the transcriptome of seeds during 1 and 3 days of alternating temperature and germinated seeds were compared with seeds incubated at constant temperature. Statistical analysis revealed that 597, 1,491, and 1,329 genes were differentially expressed (P<0.05) for the comparisons of 21-day C vs. 21-day C+1-day A, 21-day C vs. 21-day C+3-day A, and 21-day C vs. 21-day C+Germ (germination), respectively. Functional classifications based on gene set and sub-network enrichment analysis were performed to identify pathways and gene sub-networks that underlie transcriptome changes in the seeds as they germinate. Sugars, plant hormones, photomorphogenesis, and reactive oxygen species were overrepresented at 21-day C+1-day A. At 21-day C+3-day A, an increase in cellular activities was observed as the number of overrepresented pathways greatly increased. Many of the metabolic pathways were involved in the biosynthesis of amino acids, macromolecules, and energy and carbon skeleton production for subsequent germination. The 21-day C+3-day A and 21-day C+Germ pathways and sub-networks were similar and included an overrepresentation of the amino acid biosynthetic pathways; however, 21-day C+Germ seeds have an even wider array of cellular activities such as translation-related pathways, which are most likely for seedling growth. RT-qPCR analysis indicated that the up- and down-regulation of HISTONE H3, GASA2, DREBIII-1, CHS, AOS, PIF3, PLD alpha 1, and LEA may be germination-related since their expression was dramatically changed only in the 21-day C+Germ seeds. Finally, both short-term alternating temperature and short-term light exposure up-regulated the expression targets of the central hub HY5 in leafy spurge and Arabidopsis, respectively, indicating that a signaling network involving HY5 is important for germination.