Alfalfa (Medicago sativa L.) yield is severely compromised by soil salinity, especially at the level of seedling establishment. This question was addressed by proteomics to decipher whether specific changes in protein accumulation correlate with germination performance of alfalfa seeds submitted to a salinity stress as obtained by imbibing seeds in the presence of NaCl. This study used alfalfa seeds submitted to an osmopriming invigoration treatment that proved very efficient in counteracting the negative effect of salinity stress on germination performance. Comparative proteomic analyses disclosed 94 proteins commonly characterizing the response of both the untreated control and osmoprimed seeds to the experimental salinity stress. Remarkably, many of them, representing 84 proteins, showed contrasting accumulation patterns when comparing the untreated control and osmoprimed seeds submitted to the same salt stress. Thus numerous changes observed in the proteome of the untreated control seeds imbibed in the presence of salt, and presumably accounting for the loss in seed vigour associated with salinity stress, can be substantially reversed in osmoprimed seeds undergoing this stress. These data therefore provide a biochemical understanding of the increase in seed vigour generally observed with primed seeds.

The present study was conducted to evaluate the contribution of nitric oxide (NO) to the priming effects on seed germination, seedling vigour and low-temperature tolerance of two tomato cultivars (Solanum lycopersicum, cv. Cherry and Falcato) under four different constant temperatures (10, 15, 20 and 25°C). Treatments included priming in a polyethylene glycol 6000 (PEG 6000) solution (osmopriming) or presoaking in sodium nitroprusside (SNP) solution, a donor of NO, for 24 h at 25°C. Suboptimal temperatures reduced seed germination and subsequent seedling growth, as well as amylase activity, in both cultivars, but Falcato was more sensitive to low temperature than Cherry. Both osmopriming and SNP treatments enhanced germination capacity, germination index, seedling root and shoot length, α- and β-amylase activities and sugar content under suboptimal temperatures, especially in Falcato. We observed similarities between the modes of action of the two priming treatments. Both treatments increased seed NO content, whereas their positive effects on germination and subsequent seedling growth under low-temperature stress was reversed by the addition of methylene blue (MB), a scavenger of NO. This is the first report that suggests that positive effects of priming are most likely mediated through NO signalling.

Loss of vigour in leek (Allium porrum L.) seed lots is accompanied by the appearance of damage to ribosomal RNA in quiescent embryo tissue. Polyethylene glycol osmopriming treatments of such low-vigour seed permit replacement of this damaged ribosomal rRNA over a 7-day priming period. Low-vigour leek seeds germinated for 6 days without a prior osmopriming treatment still exhibit evidence of damaged ribosomal RNA in embryo tissue. Osmoprimed leek seeds dried back whilst still in the desiccation-tolerant state retain most of the benefits conferred by priming treatments. Osmoprimed leek seeds dried back when having reached the desiccation-sensitive state germinate poorly, if at all, and exhibit much reduced rates of protein synthesis in embryo tissue upon rehydration compared to desiccation-tolerant seeds. Embryo tissue from dried back, desiccation-tolerant seeds exhibits the capacity for much higher levels of protein synthesis than embryo tissue from unprimed seeds at equivalent stages of imbibition. RNA levels continue to increase in embryo tissue upon rehydration of dried back, desiccation-tolerant leek seeds but not in desiccation-sensitive seeds. Loss of protein synthetic capacity in desiccation-sensitive seeds during rehydration is accompanied by increasing levels of ribosomal RNA degradation. Such ribosomal RNA degradation may be indicative of the germinative capacity of leek seeds dried back after osmopriming treatments.

An osmotic priming treatment of 14 days in a −1.0 MPa polyethylene glycol solution improves the germination performance of a highvigour seed lot of leek (Allium porrum L., cv. Verina). Using in vivo pulse-labelling, two-dimensional polyacrylamide gel electrophoresis and fluorography, five polypeptides were found to be synthesized in embryonic tissue at 14 days of priming that were not present at 4 days of germination without prior priming. These polypeptides were still synthesized at 6 h of germination following priming. This time point lies in the 6–12 h lag phase in protein synthesis previously observed in leek embryo tissue during germination of primed seeds where there is little increase in the rate of synthesis over that seen at the end of priming. None of the polypeptides was synthesized at 2 days of germination after priming, a period of seedling growth. The five polypeptides appear to be specifically associated with the priming period. Two additional polypeptides were found in leek embryos that were synthesized at higher levels at the end of priming than during germination alone. These continued to be synthesized, at lower levels in leek embryo tissue upon germination after priming. Several polypeptides were identified in leek endosperm tissue which were synthesized at higher levels during priming than during germination and also two polypeptides whose synthesis appeared to be specific to germination.

Profiles of proteins synthesized in vivo in radicle tips were compared among unaged, artificially aged (controlled deterioration: 10% moisture content at 42°C for 3 weeks), osmoprimed (−1.5 MPa polyethylene glycol 6000 at 20°C for 1 week), and artificially aged and subsequently osmoprimed cauliflower (Brassica oleracea L.) seeds. Germination and initiation of incorporation of 35S-methionine into radicle tips were delayed by controlled deterioration. Osmopriming accelerated these processes and also alleviated the delays. The labelled proteins were analysed by two-dimensional gel electrophoresis and fluorography. Proteins were found whose expression correlated with the rate of germination, and was reduced by controlled deterioration but enhanced by osmopriming. Subsequent osmopriming reversed the effect of controlled deterioration and even caused an enhancement up to the level of unaged osmoprimed seeds. These proteins appeared to be related to processes preceding visible germination. In unaged seeds, formation of the proteins in the radicle tip began upon imbibition but decreased towards visible germination. On silver-stained gels, one of the proteins was shown to be mobilized by the time of radicle protrusion, while all were found in dry seeds. Proteins whose pronounced expression was observed only after both treatments were also found.

Osmopriming of leek seeds (Allium porrum L.) in PEG 6000 solution (−10 bars) at 15°C for 14 days leads to reductions in both the spread of germination and mean time to germination, especially in low-vigour seed lots. In vivo methyl [3H]-thymidine pulse-labelling studies have demonstrated constant and low levels of DNA synthesis in leek embryo tissue during the osmopriming treatment. DNA synthesis during osmopriming was not inhibited by aphidicolin, an inhibitor of nuclear DNA replication. Replicative and repair-type DNA synthesis was investigated using BND-cellulose chromatography and these studies revealed that about 30% of the DNA synthesis after 1 day of priming was of a repair-type. DNA repair-type synthesis contributed to approximately 20% of the [3H]thymidine incorporated into DNA during the rest of the priming period in embryo tissue from both high-vigour and low-vigour seed lots. After 1 day of germination following priming, enhanced levels of both replicative and repair-type DNA synthesis were demonstrated. The replicative type of DNA synthesis detected in leek embyros during the priming period was not inhibited by aphidicolin and appears to represent a significant level of mitochondrial DNA synthesis. DNA synthesis could be detected in both nuclei and mitochondria of leek embryo tissue during the osmopriming treatment in the absence of any detectable cell division.

A partial amino acid sequence of a vigour-related protein, V-1, from cauliflower (Brassica oleracea L.) seeds was obtained, after isolation by two-dimensional gel electrophoresis and gas-phase micro-sequencing. The sequence was found to have high homology to soybean seed maturation proteins. However, the position of the sequence in the V-1 polypeptide suggests that the V-1 and soybean proteins have different polypeptide structures. An osmoprimed seeds cDNA library was constructed and has been screened by a cDNA probe derived from the V-1 sequence. Two cDNA clones with high homology to aspartic protease (EC 3.4.23) from barley grain were isolated. The expression of putative aspartic protease mRNA was found to be enhanced by osmopriming, however the clones were found to have no significant sequence homology to the V-1.

Seed respiration has been utilized as an index of seed vigour and has correlated well with seedling development. We examined the respiration process as affected by temperature and osmopriming during imbibition in two cultivars of sorghum [Sorghum bicolor (L.) Moench] differing in capacity to germinate at low temperatures. Respiration of imbibing seeds was measured with a specially built apparatus linked to an infrared gas analyser. Seed respiration at the beginning of the imbibition process strictly depended on temperature (T). T also greatly affected the total CO2 respired to reach the first germination event, which significantly increased as T declined from the optimum. Osmopriming affected seed respiration differently in the two cultivars. In the cold-tolerant cultivar (Brandes), osmopriming reduced the total thermal requirements and, at T close to the minimum for germination, the energy required to achieve the first radicle protrusion. In the cold-sensitive cultivar (H128), osmopriming lowered base T for respiration and reduced the requirements for total CO2 respired, allowing germination even at 10°C. The cold-tolerant cultivar always maintained higher respiration rates than the cold-sensitive one, showing a greater capability of mobilizing energy from reserves. Germination time (i.e. GR50) related well to cumulative seed respiration down to a threshold value of cumulative CO2, after which GR50 was more affected by T than was cumulative respiration. Seed water uptake significantly slowed with decreasing T, and was accelerated by osmopriming. However, water imbibition rate influenced the start of seed respiration, but not the subsequent rate of metabolic activity preceding germination. Seed respiration was a good indicator of the activation of germinative metabolic processes, although under extreme T conditions the seed populations still respired, even if at low levels, but did not germinate rapidly or completely.

Abstract In seed formation the role of ethylene has received little attention. The data available on zygotic embryogenesis suggest an association of the ethylene biosynthetic pathway and seed maturation. Over the course of dicot embryogenesis, ACC-oxidase mRNA can be expressed in the cotyledons and embryonic axis. However, as maturation proceeds, cotyledonary ACC-oxidase expression disappears. In some seeds that develop primary dormancy, ethylene synthesis can be among the prerequisites for breaking dormancy. Moreover, the persistence of dormancy may be related to the difficulty of the embryonic axis to produce the necessary ethylene levels or to low tissue sensitivity. The use of inhibitors of ethylene biosynthesis or its action has provided data implicating an ethylene requirement for seed dormancy or germination in some species. However, the role of ethylene in germination remains controversial. Some authors hold that gas production is a consequence of the germination process, while others contend that ethylene production is a requirement for germination. Furthermore, among seeds that require ethylene, some are extremely sensitive to the gas, while others require relatively high levels to trigger germination. Recent studies with Xanthium pennsylvanicum seeds suggest that β-cyanoalanine-synthase is involved in ethylene-dependent germination. In addition, regulation of the partitioning of S-adenosyl-L-methionine (AdoMet) between the ethylene vs polyamine biosynthetic pathways may be a way of controlling germination in some seeds. Such regulation may also apply to the reversal of seed thermoinhibition, which can occur when polyamine synthesis is inhibited, thereby strongly channelling AdoMet towards ethylene. The biological models and approaches that may shed additional light on the role of ethylene during seed germination are presented.

Priming treatment of sunflower (Helianthus annuus L., cv Briosol) seeds for 7 days at 15°C with a polyethylene glycol solution at −2.0 MPa strongly improved their subsequent germination at 15°C on water. This stimulatory effect of priming remained after drying back the seeds at 20°C for 3 days. Malondialdehyde (MDA) and activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were measured in control unprimed seeds, primed seeds, seeds primed then dried, and after 3 and 6 hours of imbibition of controland dried primed seeds in order to determine whether the cell antioxidant systems were involved in the beneficial effect of priming. The osmotreatment resulted in a strong increase in SOD and CAT activities but did not markedly affect MDA and GR activity. Following the 3 days of drying, MDA increased and the enzyme activities became similar to those measured in dry unprimed seeds, although the stimulatory effect of priming on germination remained. Imbibition of control dry seeds was associated with an increase in MDA and a decrease in CAT and GR activities, whereas reimbibition of dried primed seeds resulted in a decrease in MDA and an increase in SOD, CAT and GR activities. Isoform patterns on native gels showed no difference between treated (priming with or without subsequent drying) and control seeds for SOD (7 isoforms) and GR (5 isoforms), but the osmotreatment did induce a second isoform of CAT. The results obtained indicate that the CAT isoform pattern might be used as an indicato of the priming treatment that promotes germination. Involvement of theantioxidant systems in seed vigour is discussed