**10. Holistic "omic" approaches of seed priming**

The identification of biomarkers of seed priming effect is a relevant goal for plant physiologists. The sequence of events associated with water-based priming involves a limited hydration step during the soaking period followed by a more or less rapid dehydration phase (**Figure 1**). It may be considered that each phase has its own profile of activation/deactivation. Transcription is not necessarily coordinately associated with translation, resulting in some cases in a limited correspondence between mRNA levels and protein abundance. Some proteins synthesized during incubation may also be degraded during dehydration, although the rate of degradation might somewhat be influenced by the rate of drying. Finally, protein synthesis may also occur as a result of the translation of long-lived mRNA previously synthesized during seed matu‐ ration [17, 37, 103, 110].

The advantage of these complementary holistic approaches is that they provide a large set of data allowing physiologist to obtain a global view of all parameters of the metabolism associated with priming. The major disadvantage, however, is that these techniques are rather expensive. As a consequence, samples are frequently analyzed after a given duration of treatment and the time-dependent evolution of parameters are rarely considered. It is still difficult to reconstitute a kinetic approach, but published studies rather provide a large set of information at a given moment while the priming procedure is a dynamic process.

#### **10.1. Transcriptomics**

Transcriptomics is the study of the transcriptome—the complete set of RNA transcripts that are produced by the genome under specific circumstances—using high-throughput methods. Several techniques are available for transcriptomic approaches, including microarray, c-DNA amplified fragment length polymorphism (cDNA-AFLP), expressed sequence tag sequencing, serial analysis of gene expression, RNAseq, and massive parallel signature sequencing [176]. According to Buitink et al. [109], more than 1300 genes may be differentially regulated during priming with PEG at -1.7 MPa in *Medicago truncatula*. Genes whose expression are regulated during the priming process are commonly categorized according to the function of the corresponding protein (metabolism and regulation of metabolism, cell cycle regulation, DNA processing, transcription regulation, cellular transport and communications, stress responses, etc.). However, a consistent part of the identified genes is still not annotated.

In *Brassica napus*, numerous priming-regulated genes are involved in gluconeogenesis, which is essential for triacylglycerol breakdown into small molecules. Another important category of genes involved in water-based priming of rapeseed encodes for transcription factors, and this is especially the case after hormopriming with ABA [138]. In the same species, it was demonstrated that germination of primed seeds involves a specific set of genes comparatively to germination of unprimed ones [36]. In young seedlings obtained from primed seeds, the expression of stress-related genes is often more rapid than in plants from unprimed ones, as recently exemplified for cold tolerance [26]. Not only genes related to protein synthesis but also genes involved in protein degradation may be induced by priming. Some transcription factors may be down-regulated during soaking and up-regulated during drying [36]. Genes involved in the synthesis of osmoprotectant, such as proline, may also be regulated at various steps of priming [120].

In *Brassica oleracea*, transcripts that are abundant in dry seeds rapidly decline during osmo‐ priming and germination expression programs are initiated during osmopriming [138]. For these authors, genes expressed during slow drying following the soaking period are correlated with the stress resistance properties of primed-material. This may especially concern cell-cycleregulated genes, enzymes involved in C metabolism, and components of the translation machinery. Acquisition of the desiccation tolerance after the soaking period is a crucial trait for priming. Transcriptomic data demonstrated that the molecular determinism associated with such trait may be, at least partly, similar to those involved during a « normal » seed dehydration process during maturation. In *Medicago truncatula*, several regulatory genes expressed during drought stress are also up-regulated during seed maturation. During priming, these genes are involved in post-soaking dehydration while genes conditioning cell cycle, cell wall biogenesis, and energy metabolism are repressed [109]. Transcripts accumu‐ lated after PEG-priming treatment comprise those encoding for LEA and seed storage proteins, or genes controlling seed dormancy while genes involved in photosynthesis and cell wall modifications are commonly repressed [108].

Beside involvement of transcription factors, it could be of primary importance in the future to analyze the priming-induced epigenetic changes. Indeed, DNA methylation may be directly involved in « stress memory » and some changes, such as cytosine methylation or overex‐ pression of histone deacetylase was reported to occur during germination [78].

#### **10.2. Proteomics**

The advantage of these complementary holistic approaches is that they provide a large set of data allowing physiologist to obtain a global view of all parameters of the metabolism associated with priming. The major disadvantage, however, is that these techniques are rather expensive. As a consequence, samples are frequently analyzed after a given duration of treatment and the time-dependent evolution of parameters are rarely considered. It is still difficult to reconstitute a kinetic approach, but published studies rather provide a large set of

Transcriptomics is the study of the transcriptome—the complete set of RNA transcripts that are produced by the genome under specific circumstances—using high-throughput methods. Several techniques are available for transcriptomic approaches, including microarray, c-DNA amplified fragment length polymorphism (cDNA-AFLP), expressed sequence tag sequencing, serial analysis of gene expression, RNAseq, and massive parallel signature sequencing [176]. According to Buitink et al. [109], more than 1300 genes may be differentially regulated during priming with PEG at -1.7 MPa in *Medicago truncatula*. Genes whose expression are regulated during the priming process are commonly categorized according to the function of the corresponding protein (metabolism and regulation of metabolism, cell cycle regulation, DNA processing, transcription regulation, cellular transport and communications, stress responses,

In *Brassica napus*, numerous priming-regulated genes are involved in gluconeogenesis, which is essential for triacylglycerol breakdown into small molecules. Another important category of genes involved in water-based priming of rapeseed encodes for transcription factors, and this is especially the case after hormopriming with ABA [138]. In the same species, it was demonstrated that germination of primed seeds involves a specific set of genes comparatively to germination of unprimed ones [36]. In young seedlings obtained from primed seeds, the expression of stress-related genes is often more rapid than in plants from unprimed ones, as recently exemplified for cold tolerance [26]. Not only genes related to protein synthesis but also genes involved in protein degradation may be induced by priming. Some transcription factors may be down-regulated during soaking and up-regulated during drying [36]. Genes involved in the synthesis of osmoprotectant, such as proline, may also be regulated at various

In *Brassica oleracea*, transcripts that are abundant in dry seeds rapidly decline during osmo‐ priming and germination expression programs are initiated during osmopriming [138]. For these authors, genes expressed during slow drying following the soaking period are correlated with the stress resistance properties of primed-material. This may especially concern cell-cycleregulated genes, enzymes involved in C metabolism, and components of the translation machinery. Acquisition of the desiccation tolerance after the soaking period is a crucial trait for priming. Transcriptomic data demonstrated that the molecular determinism associated with such trait may be, at least partly, similar to those involved during a « normal » seed dehydration process during maturation. In *Medicago truncatula*, several regulatory genes expressed during drought stress are also up-regulated during seed maturation. During

information at a given moment while the priming procedure is a dynamic process.

24 New Challenges in Seed Biology - Basic and Translational Research Driving Seed Technology

etc.). However, a consistent part of the identified genes is still not annotated.

**10.1. Transcriptomics**

steps of priming [120].

Proteome is the entire set of proteins present at a given moment in a given biological sample and proteomic is the large-scale study allowing identification and quantification of these proteins. Proteomics has now been practiced in different plant systems through the separation of proteins by two-dimensional gel electrophoresis followed by peptide mass fingerprinting. Tandem mass spectrometry can get sequence information from individual isolated peptides. Quantitative proteomics represent an important extension enabling the comparison of changes in protein levels across different samples [177]. Gel-free shotgun proteomic is an alternative for identification and quantification of protein in large-scale studies. This method was recently used to reveal potential biomarkers of priming-induced salt tolerance in durum wheat with a concomitant use of protein fractionation and hydrogel nanoparticles enrichment technique [27]. According to these authors, hydropriming was accompanied by a significant change in 72 proteins. Most of them are involved in proteolysis, protein synthesis, metabolism regulation, and disease or defense response. Priming with ascorbic acid changed the pattern of proteome signature and most of identified proteins are involved in metabolism regulation, antioxidant protection, repair processes, and methionine-related metabolism.

Cycloheximide, a potent inhibitor of protein synthesis, may be added to priming or to germination solution to analyze the precise requirement of protein synthesis during these steps. It appears that protein synthesis is not necessarily required for normal germination process, which could be related to the fact that in some cases, germination relies only on translation of pre-stored long-lived mRNA produced during seed maturation [14, 178]. Cycloheximide may drastically affect osmopriming-induced improvement of Cd resistance in *Poa pratensis* and *Trifolium repens* in relation to a decrease in α and β-amylase [179]. Protein concentration in primed seeds is the result of two contrasting pathways: the first implies specific protein synthesis while the second involves the breakdown of storage proteins through protease activation. It was demonstrated that priming may act on protease activities and also influence the expression of protease encoding-gene [180].

Some strategies are also used to separately identify proteins in embryos and in embryosurrounding tissues. In monocots, a special attention is often paid to aleurone layers involved in hydrolytic enzyme synthesis [181]. Aleurone layer is easily separated in some plant material such as wheat or barley but is more difficult with other monocot such as turfgrasses with very small seeds. In wheat, it was demonstrated that the set of proteins regulated by priming is quite different in embryos and in surrounding tissues and the number of proteins affected by priming was by far higher in the embryos than in the other parts of the seed. Proteins regulated in embryos involve those directly linked to metabolism regulation (especially methionine biosynthesis, glutamate/glutamine metabolism, amino acids synthesis, etc.), energy supply, cell growth and maintenance of cell structure while proteins up-regulated in surrounding tissues are mainly involved in reserves mobilization or in the management of the oxidative stress [67]. In barley, however, ascorbate peroxidase was observed only in the embryo while several other redox-related proteins differed in spatio-temporal patterns at the onset of radicle elongation [181].

Priming is considered as an invigoration treatment and several proteomic approaches were performed to unravel the biomarker of seed vigor. Catusse et al. [182] found that 18 proteins are accumulated during hydropriming of sugarbeet seeds and that the same proteins appear directly reduced during aging. Seed vigor appears directly related to lipid and starch mobili‐ zation, protein synthesis and methionine cycle. In poplar, more than 81 proteins showed a significant change in abundance when comparing the proteomes among seed with different vigor [183]. According to these data, the decrease in seed vigor is an energy-dependent process, which requires protein synthesis and degradation as well as cellular defense and rescue. Salicylic acid (SA) was proposed as an invigorating elicitor promoting seed germination under saline conditions: SA re-induced the late maturation program during early stages of germi‐ nation, induced the synthesis of antioxidant enzymes, and improved the quality of protein translation [184]. Similarly, the "prime-ome" approach performed by Tanou et al. [185] confirmed the importance of redox proteomic and processes such as N-nitrosilation, tyrosine nitration, and mitogen-activated protein kinase MPK3 signaling in priming effects.

Osmotic priming was reported to trigger desiccation tolerance in *Medicago truncatula* [110]. Proteomic analysis demonstrated that such trait is directly linked to the synthesis of lateembryogenesis abundant proteins from different groups. Secondary structure of some proteins was compared in the hydrated and dry state after fast or slow drying using Fourier transform infrared spectroscopy, which confirms that these proteins adopted α helices and β-sheets conformation during drying process.

Some proteomic approaches are conducted on plant species whose genomes are not se‐ quenced. A recent 2DE-MS/MS-based proteomic study was conducted on pearl millet seeds primed by β-aminobutyric acid and showed an over-representation of proteins belonging to glucose metabolism, and a majority of induced proteins are directly related to energy [106]. Seedlings issued from those seeds are more resistant to downy mildew (*Sclerospora graminico‐ la*). It is interesting to note that several of the elicited proteins are present in the extracellular space and in organelles (mainly mitochondrion and chloroplast).

#### **10.3. Metabolomics**

Metabolome refers to the complete set of small-molecule metabolites present within a plant tissue or organ at a moment. Metabolomic should thus be considered as the quantitative measurements of the whole set of compounds involved in the metabolism of a given biological sample. Plant metabolomics has become an essential part of functional genomics. It often appears difficult to analyze the entire range of metabolites by a single analytical method, and several tools are thus commonly combined for this purpose: high performance liquid chro‐ matography (HPLC), gas chromatography (GC), electrospray ionization coupled with mass spectrometry, capillary electrophoresis, atmospheric pressure chemical ionization (APCI), and secondary ion mass spectrometry (SIMS). Metabolomic approach, however, still remains difficult to perform on seed material considering the high proportion of molecule issued from reserve mobilization. An excess of soluble sugar in graminaea and an excess of small lipids issued from oil digestion in oleaginous plant material may greatly hamper isolation of other compounds involved in hastening the germination of primed seeds. Similarly, some important compounds might be present only in specific tissues or cell compartments and react with sugars during extraction processes; this is especially the case of gibberellins which play a crucial role in germination but are believed to conjugate with sugar or phenol compounds, which greatly compromise isolation of and identification procedures of numerous metabolites [186].

Priming process may drastically modify the synthesis and accumulation of endogenous antioxidant such as glutathione, ascorbic acid, and even α-tocopherol. A modification in reducing sugars concentration, such as glucose resulting from partial starch digestion during priming, may influence protein glycation, a nonenzymatic reaction between reducing sugars and amino groups in protein [150]. The amount of proline was reported to be modified in osmoprimed seeds [120]. Some other data reported modifications in aspartate, leucine, threonate, glutamate, fumarate, or pinitol content in primed seeds from different species [1, 2, 35, 104, 152]. Methionine may also play a key role in priming process, mainly as a precursor of both ethylene and polyamine synthesis. Polyamines are small aliphatic molecules influenc‐ ing all aspects of plant metabolism and development, including the germination processes. Ethylene is also involved in various aspects of seed germination, and priming was reported to modify the kinetics of ethylene synthesis from its precursor 1-aminocyclopropane-1 carboxylic acid (ACC) [186]. Other phytohormones are expected to assume important func‐ tions in priming effect (ABA, gibberellins, cytokinins, auxin, etc.) [50, 52], but the quantitative impact of priming procedure on these compounds is still not well established.
