**11. Main limitations of priming techniques**

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

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

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

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

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

space and in organelles (mainly mitochondrion and chloroplast).

elongation [181].

conformation during drying process.

**10.3. Metabolomics**

Seed priming has emerged as an effective approach for increasing seed vigor. The optimal treatment differs between species, cultivar, and seed lots. Such variability is a major limitation of the priming method since numerous trials are required to identify the most appropriate strategy for each situation. There is no "general rule" concerning modalities of seed priming and there is no clear trend of priming response according to the taxonomic position of the species [188]. This, undoubtedly, limits the commercial implementation of priming treatments.

Some priming treatments may imply a risk of medium contamination by fungi and bacteria, which may heavily impair subsequent seed germination [189]. This may especially be the case for PEG and sometimes requires the simultaneous use of pesticide, although the impact of these compounds on the priming efficiency remains unknown. After priming treatment, seeds are dried back to their initial moisture content, but this dehydration phase is usually performed rapidly and occurs faster than classical dehydration of maturing seeds. It has been hypothe‐ sized that this brutal desiccation procedure alters the beneficial effect of priming [2].

The major drawback of priming is that it reduces the longevity of primed seeds as compared with the nonprimed seeds [190–192]. Storability of primed seed material is consequently reduced, and this results in higher costs for material maintenance for farmers and seed companies. The loss of viability, however, appears quite variable depending on the species, cultivars, and seed lots. In extreme cases, priming-induced advantages may even disappear after only 14 d of storage and the obtained seedling may then perform worse than those issued from unprimed seeds [193]. One of the limitations of these studies, however, is that experi‐ ments are commonly performed on artificially aged seeds using short-term exposure to high temperature in a moist environment, which is not necessarily fulling relevant from a real aging process. Some studies, using classical long-term strategies, reported that longevity is not necessarily affected or may even be increased by priming treatments [194]. Hussain et al. [195] recently demonstrated that post-priming temperature plays a key role in maintenance of seed longevity, which is indeed rapidly compromised in rice seeds maintained at room temperature while it remains intact when the material is stored at 4°C. According to these authors, the deleterious effect observed at 25°C storage was related to hampered starch metabolism in primed seeds. It was also suggested that maintenance of the seed longevity at 4°C may be due to a high viscosity that strongly reduces molecular mobility in the cytoplasm and thus limits the impact of deteriorative process even in seeds exhibiting low water content [196]. Oxygen during storage may trigger metabolic processes in primed seeds, which did not re-establish a true quiescent stage after dehydration when stored at room temperature while oxygen had no influence at low temperatures [195].

In some cases, repeat priming treatment after storage may partly remove the damaging effect on seed viability [193] while, in other cases, such a loss is permanent and not reversible [194]. Whatever, the fact that an additional treatment may be required to restore full germination potential represents an additional cost and source of variability.
