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Seed dormancy is an important stage in the life cycle of many non-domesticated plants, often characterized by the temporary failure to germinate under conditions that normally favor the process. Pre-treating dormant imbibed seeds at a constant temperate accelerated germination of leafy spurge seeds under alternating temperatures. However, dormant seeds will also germinate without a pre-treatment, albeit at a much slower rate, which gives rise to longer periods of imbibition before germination. Transcriptome analyses on seeds exposed to prolonged imbibition highlighted pathways associated with phenylpropanoid biosynthesis and interacting networks of genes involved in plant defense. In addition to the many pathways associated with phenylpropanoid biosynthesis enriched with down-regulated genes upon germination, there were also numerous pathways enriched with up-regulated genes associated with energy metabolism, such as glycolysis. Transcriptome data further suggest that metabolism and signaling by the plant hormones ethylene, gibberellin, and abscisic acid are involved in the developmental transition from dormancy to germination. More specifically, sub-network enrichment analysis identified ABI3 as a central hub of a sub-network at germination including several down-regulated genes such as DELLA (i.e., RGL2), which represses gibberellin signaling processes required for germination. The 595-fold increase in the expression of ACC oxidase (ACO4) at germination also suggests an important role for ethylene biosynthesis in germinating leafy surge seeds. Furthermore, the 10-578-fold difference in expression of many genes such as HY5 and Histone H3 between two populations at germination, which were treated with and without a constant temperature germination-enhancing pretreatment, revealed disparate impacts on various biosynthetic, growth, signaling, and response processes. Overall, our results indicate a constant temperature pretreatment (20°C for 21d) is not required for germination of leafy spurge seeds at an alternating temperature. However, the presence or absence of the pretreatment does affect the rate of germination and the germination transcriptional programs., (Published by Elsevier GmbH.)

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 α1, 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.

We investigated transcriptome changes in Euphorbia esula (leafy spurge) seeds with a focus on the effect of constant and diurnal fluctuating temperature on dormancy and germination. Leafy spurge seeds do not germinate when incubated for 21 days at 20 degrees C constant temperatures, but nearly 30% germinate after 21 days under fluctuating temperatures 20:30 degrees C (16:8 h). Incubation at 20 degrees C for 21 days followed by 20:30 degrees C resulted in approximately 63% germination in about 10 days. A cDNA microarray representing approximately 22,000 unique sequences was used to profile transcriptome changes in the first day after transfer of seeds from constant to alternating temperature conditions. Functional classification based on MIPS and gene ontology revealed active metabolism including up-regulation of energy, protein synthesis, and signal transduction processes. Down-regulated processes included translation elongation, translation, and some biosynthetic processes. Subnetwork analysis identified genes involved in abscisic acid, sugar, and circadian clock signaling as key regulators of physiological activity in seeds soon after the transfer to alternating conditions.

Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP- like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis "general purpose" traditional reference genes (GAPDH&lowbar;1, GAPDH&lowbar;2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the CT values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ΔCT method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes from other plant species. [ABSTRACT FROM AUTHOR]