FR2748481A1 - Seed protein specifically expressed during pre-germination stages - Google Patents

Abstract

Isolated plant protein (A) is expressed specifically in seeds. It contains \- 1 18-25 kDa unit and appears during the pre-germination (priming) stage. Also claimed are: (1) proteins (B) equivalent to (A); (2) mono- and poly-clonal antibodies (Ab) that recognise \- 1 epitope on (A) or (B), and (3) molecular probes (protein or nucleic acid) derived from (A) or (B), or nucleic acid encoding them, or from Ab.

Description

Protein usable as a marker for the imbibition of a semen during
germination
The present invention relates to a process for obtaining and a new protein induced during the imbibition of mature seeds of plants belonging to species of the families of chenopodiaceae, composed, labiated and cucurbits and its use as a molecular marker of the phase of imbibition involved in the germination of these seeds and in the osmo- and hydro-conditioning processes known as pre-germination of these seeds.

 Germination is a complex development process for which there is currently little precise molecular data. This development program, during which the cells of the embryo pass from a state of rest to a state of intense metabolic activity, is implemented essentially during the imbibition phase. It ends, in the physiological sense, with the piercing of an organ of the budding seedling through the envelopes of the seed (Bewley & Black, 1983).

 The main techniques used to define the competence of seeds to germinate use conventional germination tests, that is to say that we measure, for a given seed batch and under codified conditions (temperature, humidity, light, substrate), the percentage of germination at different times after sowing. The criterion generally used to quantify germination is the breakthrough of the seed coat by an organ of the budding plant. Most of the biochemical markers described to date are correlated with this phase. They are therefore not sensu stricto markers of germination, but rather of the initial stages of growth (Fincher, 1989). It should be noted that the initial phase of imbibition (ie water intake) is reversible up to a certain point. Following controlled hydration of the seeds, they can be dried, while retaining their biological integrity and their germination capacity. As soon as the seedling appears, its engagement in its growth becomes irreversible. Indeed, dehydration at this stage irreparably causes the death of the seedlings (Bewley & Black, 1983).

The pre-germination processes developed by seed companies are based on the reversibility of the initial soaking phase. The seeds are generally hydrated in a controlled manner, then they are dried (Coolbear et al., 1987;
Karsen et al., 1989; Tarquis & Bradford, 1992). These processes bring real added value to seeds because they allow:
1) to homogenize the seed lots in relation to germination,
2) an appreciable saving of time for emergence after sowing, because a certain number of biochemical processes necessary for the achievement of germination would already be carried out during the treatment,
3) an improvement in the germination quality of older seed lots, probably due to the fact that mechanisms for repairing damaged biological structures during the final maturation of seeds, or even during conservation, are put in place during of treatment.

 At present, the optimization of such processes is based solely on the conduct of germination tests, which require several days of experimentation. In addition, if the treatment fails (the seed batches being by nature heterogeneous, it is therefore necessary to optimize the treatment for each of the batches), the batch is lost. The possibility of continuously monitoring the imbibition phase, via an easy-to-detect molecular marker, would therefore constitute considerable progress, making it possible to adapt the pregermination treatment to each batch of seeds.

 The subject of the present invention is a protein of plant origin, induced during the imbibition of seeds, characterized in that it comes from a species of plant culture, that it comprises at least one unit from 18 to 25 kDa, is expressed in seeds and in no other organ of the plant, and appears intensely during the early stages of germination (imbibition) and during pre-germination processes using a water intake controlled by seeds.

 The present invention relates more particularly to a pure protein, obtained from seeds soaked in chenopodiaceae, for example beet, spinach and lamb's quarters, but also in other families such as for example compounds such as for example chicory and the bitterness, the labiates such as for example the lamier, or even the cucurbits such as for example the zucchini. In the case of beets, the protein is called SIP.

 The invention also relates to new antibodies, called anti-SIP, characterized in that they recognize the beet SIP protein and the proteins equivalent to the latter in the seeds of the various families mentioned above.

 It also relates to the use of the specificity of tissue expression and of the pattern of development of these markers in order to measure as precisely as possible the progress of the imbibition of the seeds, either during germination or during pre-germination processes of seeds, in particular their use as a molecular marker of the imbibition of seeds of chenopodiaceae, for example beet and spinach, but also in other families such as for example compounds such as by example chicory and bitterness, labiatae such as for example the lamier, or cucurbits such as for example zucchini. The detection of these markers is carried out with specific anti-SIP antibodies.

 This detection can be done using a device (kit), which is also part of the invention.

Characteristics of a protein induced by imbibition of seeds during pre-germination processes:
Sugar beet seed (Beta vulgaris, Universe variety) and two known pre-germination techniques are used as a study model. The seeds used have not undergone any prior treatment, their average hydration rate is around 12%.

The pre-germination techniques used are on the one hand the hydro-conditioning of seeds 9 at low temperature and in the presence of pure water (Coolbear et al., 1987) and on the other hand the osmo-conditioning of seeds in presence of an inert osmoticum, polyethylene glycol (PEG) 6000 (Tarquis & Bradford, 1992).

 1) Technic of D-eermination by bone by hvdro-conditlonnement at low temperature.

This technique has been described in the case of tomato seeds (Coolbear et al., 1987). Its principle consists in soaking the seeds under defined conditions, at a temperature low enough to prevent the seeds from germinating. After treatment, the seeds are dried, which allows their conservation. This method is adapted here to the case of beet seeds. The germination and pre-germination experiments are carried out in the following manner. Each experiment relates to a batch of one hundred beet seeds which are placed in Petri dishes (12 x 12 cm) on 3 layers of filter paper, soaked with 6 ml of distilled water. The seeds are covered with a sheet of filter paper which, acting as a wick, allows the seeds to soak over their entire surface, then the boxes are closed with their covers so as to minimize the evaporation of water. The boxes are then placed in temperature-controlled cabinets in the dark. The germination curves obtained at each temperature, represented by the evolution of the cumulative germination rate (expressed as a% of the total number of seeds analyzed) as a function of the germination time, are characterized by three phases. First, a latency phase, during which no germination takes place and during which the seed is hydrated. The duration of this latency phase is all the longer the lower the incubation temperature. It is for example from 32 h at 250C, from 44 h at 20 "C, from 65 h to 15" C, from 120 h to 10 C and from 320 h to 5 C. Second, a phase of increase in the rate of germination, the speed of which increases with the incubation temperature in the studied range which goes from 50C to 25 "C. This speed also characterizes the homogeneity of the seed lot analyzed. The more the seeds have a homogeneous germinative capacity, the more this speed is great, and vice versa. Third, reaching a plateau characterizing the maximum germination value of the seed lot considered. When the beet seeds incubated for 11 days at 5 "C under the conditions described above, and of which none have germinated, are sampled, dried at 20 "C until bringing them to a hydration rate of 10%, these seeds germinate much faster than the control seeds which have not undergone any prior treatment. For example, for seeds treated at 5 "C for 11 days, the latency time e of the germination curve at 150C is only 25 h, compared to a latency time of 65 h for the control seeds. This method, as described by Coolbear et al. (1987) for tomatoes, therefore allows effective pre-germination of beet seeds. In the case of beets, the use of low temperatures (5 "C for example) is beneficial because if, at 20 and 25" C, the hydro-conditioning is faster, it is also more difficult to control, the seeds germinates easily at high temperatures. At 50C, on the other hand, any root breakthrough is blocked for long periods of time spanning several days, while allowing controlled hydration of the seeds. There is therefore, at low temperature, a good decoupling in time between the processes of imbibition and germination. Such decoupling and intense blocking of the root breakthrough at 50C also allow better control of the subsequent dehydration of the winning seeds, without risk of deterioration of the seed lot 0 hydration is indeed reversible until the seeds germinate ). This drying step thus authorizes the conservation of the treated seeds for several months.

In parallel with these germination studies, part of the seeds are taken at different times from incubation at controlled temperature to carry out biochemical analyzes. For this, crude extracts of total and soluble proteins are produced in the following manner: grinding of the seeds in a Waring blender type mixer for 60 sec and in the presence of liquid nitrogen, taking up the powder in a homogenization buffer (Hepes pH 8.0) containing various protease inhibitors (benzamidine-HCl, phenylmethylsulfonyl fluoride, e-amino caproic acid), centrifugation (30 min at 20,000 g in Eppendorf centrifuge tubes) to remove cell debris, carrying out tests based on conventional techniques for the separation of proteins from the polyacrylamide gel extract in the presence of SDS. This latter technique has been optimized to allow the use of electrophoretic microanalysis materials for proteins in preformed polyacrylamide gels (PhastSystem and PhastTransfer from Pharmacia), which have the advantage of conducting sample analysis very quickly (electrophoresis and Protein staining of the gel with Coomassie blue is carried out in less than an hour; 16 samples are analyzed at once) and with little material (1 μl of protein sample deposited per gel track). This apparatus also performs the electrophoretic transfer of the proteins from the gel onto a nitrocellulose sheet according to the technique of
Towbin et al. (1979), which subsequently makes it possible to reveal certain of the proteins of the extract analyzed with specific antibodies ("Western blot" method).

 Using these methods, we observed the massive induction of a protein of molecular mass (22 i: 3) kDa during the hydro-conditioning of beet seeds at 50C. We name this protein SIP (for Seed Imbibition Protein). It is present at a basal level in untreated control seeds and becomes very abundant in treated seeds (5 to 7 times the basal level of controls), representing approximately 5 to 10% of the soluble proteins of pre-germinated seeds.

 The SIP protein was purified in the state of homogeneity from a protein extract (produced as described above) from pre-germinated beet seeds using the hydro-conditioning process at 50C (produced as described above). -above). The proteins of the extract are separated electrophoretically in a 20% polyacrylamide preparative gel in the presence of SDS. After electrophoresis, the SIP protein, identified according to its mass and its migration, is electroeluted from the gel according to the technique of Teissère et al. (1990). The solution is then subjected to centrifugation (6000 g) on Filtron Green, which allows the concentration of the sample and the elimination of most of the SDS.

This sample was used to immunize a rabbit (3 successive injections of 100 pg of SIP in the presence of the complete Freund's adjuvant, the first at time zero, the next two 3 weeks and 7 weeks after the first). The antibodies thus obtained (anti
SIP), which are also part of the invention, allow, at a dilution of 1/6000, the detection of approximately 5 ng of SIP protein according to conventional ELISA techniques in micro-titration plates,
2) Pre-germination technique by osmo-conditioning. This pregermination technique has been described by many authors, for example by Tarquis & Bradford (1992) in the case of lettuce seeds. Its principle consists in incubating seeds in the presence of an inert osmoticum, PEG, so as to inhibit germination, that is to say root breakthrough, while allowing hydration of seeds. Then, the seeds thus treated are dried, which allows their conservation. In the case of osmoconditioning of beet seeds, the same technique is used as for hydro-conditioning, except that the seeds (one hundred seeds per 12 x 12 cm box) are incubated at 290C in the presence of 6 ml of distilled water containing polyethylene glycol (PEG 6000). Adjusting the concentration of PEG 6000 makes it possible to obtain variable osmotic potentials (Michel & Kaufmann, 1973). The lowering of the osmotic potential leads to a progressive slowing down of the speed of germination. Thus, at -7.5, 10, -15 and -20 bars, there are no observable root breakthroughs for at least 200 h at 20 "C. The control seeds incubated in the absence of PEG 6000 germinate after a phase latency of 50 h. When the beet seeds incubated for 8 days at 20 ° C. and at -7.5 bars under the conditions described above, and none of which has germinated, are removed, washed briefly to remove the PEG 6000, dried at 20 "C until bringing them to the 10% hydration rate, these seeds germinate much faster than control seeds which have not undergone any prior treatment. For example, for seeds treated at 20 "C and -7.5 bars for 8 days, the latency time of the germination curve at 200C is less than 8 h, compared to a time of 50 h for seeds This treatment method, described for example by Tarquis & Bradford (1992) for the pre-germination of lettuce seeds, therefore also allows effective pre-germination of beet seeds.

 Using these methods, we observed the massive induction of the SIP protein during the osmo-conditioning process (8 days of soaking at 200C in the presence of PEG 6000 giving osmotic potentials of -7.5 bars, -10 bars and -15 bars).

Correlation between SIP protein induction rate and seed gnmmg intensity
The use of anti-SIP antibodies shows that the SIP protein is strongly induced (by a factor of 5 to 7) during the osmoconditioning of seeds in the presence of PEG 6000. The same is true during the hydro process -conditioning of seeds in the presence of water alone at 50C. In the latter case, the protein appears after 6.5 days of incubation under these conditions, the maximum level (approximately 6 to 7 times the basal level of untreated seeds) being reached after 11 days of incubation. The intensity of the pre-germination treatment of the seeds is characterized as follows. Samples of seeds are taken during the hydro-conditioning treatment at 50 ° C., they are dried, then they are put back to germinate at 15 ° C. We then measure, for each of the samples, the germination rate obtained at temperature 15 "C for a fixed time of 63 hours. These measurements indicate that the longer the incubation time at 5 "C, the higher the germination rate at 63 hours and 150C of the treated seeds, that is to say that the intensity of priming increases with duration. of hydro-conditioning at 50 C. The use of anti-SIP antibodies shows the existence of a positive correlation between the level of SIP synthesized during the seed treatment and the intensity of the pre-germination treatment. further demonstrate the possibility of continuously monitoring the progress of a pre-germination process through the use of ariti-SIP antibodies and a classic ELISA technique. In the case of model sugar beet (Univérs variety) , an increase by a factor of 6 to 7 in the content of seeds treated with SIP protein (compared to the basal level of control seeds) corresponds to the optimal pre-germination conditions of these seeds. When this level of SIP is reached, one must Stop the treatment, because if it is prolonged there is a risk of germinating part of the seeds, and these seeds treated too intensely will be lost during the subsequent dehydration necessary for the conservation of the treated seeds, thus causing a loss of quality of the batch of treated seeds (dehydration is not reversible after the root breakthrough).

 The kinetics of appearance and the quantification of the rate of SIP during the implementation of any method of pre-germination of seeds are also part of the invention.

Characterization of seed proteins induced during the imbibition phase of eermination
The previous results demonstrate the massive induction of the SIP protein during the pre-germination processes of beet seeds. The question is whether the induction of this protein normally accompanies germination, or whether it is established specifically in response to the pre-germination processes used. To answer this question, germination experiments are carried out at the optimum temperature for germinating beet seeds. Under the defined germination conditions (one hundred seeds per 12 x 12 cm Petri dish, 6 ml of water per dish) such optimum germination is obtained in the range of 200C to 25 "C. Under these conditions, we measure on the one hand the cumulative germination rate of lots of beet seeds at various incubation times in the thermostatically controlled cabinet, and on the other hand part of the seeds are taken at different times to quantify the rate of SIP, by carrying out crude extracts of total and soluble proteins as described above, and using anti-SIP antibodies The protein is present at a basal level in mature and dry seeds, then it is strongly induced during germination, at a good rate faster than the radicles appear. Thus, for seeds set to germinate at 20 "C, the rate of SIP is approximately multiplied by three, while no seed has yet germinated. Then, the amount of protein
SIP gradually increases to its maximum value (5 to 7 times the basal level), reaching its maximum value after 96 h at 20 "C, while the cumulative percentage of germination also increases. Finally, the SIP protein gradually disappears after 96 h of germination at this temperature, and that the growth of the seedling sets in.

 In conclusion, the SIP protein is normally induced during the imbibition and germination of beet seeds.

Tissue distribution of protein
The use of anti-SIP antibodies shows that the expression of the SIP protein is specific for seeds. It is not detected in the vegetative organs of the plant such as the leaves and roots.

Presence of proteins equivalent to the SIP protein in other seed species
We looked for the possible presence of proteins equivalent to the SIP protein in various seeds. Having observed that, in the case of sugar beet (variety
Universe), the SIP protein is induced during the early phases of germination at 20 "C, various seed lots were germinated (100 seeds per Petri dish, 6 ml of water per dish, T = 200C). Samples are taken when the batches reach 50% germination under these conditions. The use of anti-SIP antibodies shows that the protein is present in all varieties of germinated sugar beet seeds (Universe,
Adonis, Alizé, Accord), and fodder (Monoval Géant Rouge, Eckendorf) that we have analyzed, and in different goosefoot (spinach, white goosefoot), papilionaceae (lupine, bean, pea, alfalfa), compound (sunflower, chicory, honeycomb) , lettuce), labiata (lamier), cucurbits (zucchini, pickle), cruciferous (rapeseed, mustard, radish,
Arabidopsis thaliana) and nightshade (tomato). For spinach, chicory, ragwort, lamier and zucchini, the level of proteins equivalent to the SIP protein increases sharply during germination. For these seeds, the antibodies reveal one or more proteins (two for chicory, three for spinach and zucchini) whose mass is close to 20 kDa (18 to 25 kDa). It could therefore be isoforms of the SIP protein (multigenic family or post-translational modifications). For species belonging to the cereal family (barley, corn, wheat, rice) and to the umbelliferous family (carrot), the reactivity of anti-SIP antibodies is very low.

Conducting a pre-germmatlon process using a diagnostic kit
The present invention also relates to a method for optimally carrying out a pre-germination treatment of seeds which, during the treatment, synthesize a protein recognized by anti-beet SIP antibodies, such as seeds of the family of chenopodiaceae, for example beet, lamb's quarters and spinach, but also in other families such as, for example, compounds such as chicory and bitterness, labiates such as for example lamier, or cucurbits such as for example zucchini. The diagnostic kit offered consists of these specific antibodies. It is supplemented by secondary anti-rabbit antibody antibodies (anti-SIP being made in rabbits). These secondary antibodies are commercially available (e.g. Sigma for rabbit anti-antibody made in goats). They are coupled to peroxidase, allowing easy quantification of the SIP protein (if it is beet) or its equivalent (if it is seeds other than beet) in protein extracts leading to extraction of all the soluble proteins as described for beet. This quantification is carried out using a conventional ELISA method, using micro-titration plates and spectrophotometric reading of the development of the peroxidase reaction in the presence of peroxidase substrates [H202 and 2-2'-azinobis- (3-ethylbenzothiazoline- 6-sulfonic acid),
Aldrich] using a micro-titration plate reader (EL340 reader from Bio-Tek
Instruments for example).

Firstly, a germination experiment is carried out at the optimum germination temperature of the seeds considered (for example 20 to 25 ° C. for sugar beet seeds of the Universe variety), and the cumulative germination rate and the induction of protein synthesis reacting with anti antibodies
SIP. This makes it possible to determine the maximum rate (TMax) of synthesis of the protein recognized by the anti-SIP antibodies. This maximum value, TMax, will set the upper limit of the level of protein recognized by anti-SIP antibodies, not to be exceeded when implementing a pre-germination process. The pregermination treatment is then carried out, following the increase in the rate of synthesis of the protein recognized by the anti-SIP antibodies over the course of the treatment. When TMaX is reached, treatment must be stopped, because if it is prolonged there is a risk of germinating part of the seeds, and these seeds treated too intensely will be lost during the subsequent dehydration, necessary for the conservation of the treated seeds, thus leading to loss of quality of the treated seed lot.

The subject of the invention is also a process for controlling the imbibition or the water uptake during the germination of seeds, either natural or during pregermination treatment, characterized in that a SIP protein is used as molecular marker or an equivalent protein.

 The following examples illustrate the isolation of the SIP protein, the role of the SIP protein and the equivalent proteins and their use for controlling the imbibition or the uptake of water during germination of seeds either natural or natural. pre-germination treatment.

EXAMPLE 1. Evolution of the total proteins and of the SIP protein during the pregermination of the beet seeds. Isolation of the protein SIP (A) Traling of beet seeds by h conditionning at 50C
The pre-germination experiments are conducted according to the hydroconditioning process. Beet seeds are incubated for 11 days at 50C in the presence of 6 ml of water per hundred seeds. The total and soluble proteins are extracted by grinding in a Hepes buffer (pH 8.0) containing protease inhibitors. These proteins are analyzed after electrophoresis in polyacrylamide gel (20%) PhastSystem from Pharmacia in the presence of SDS and staining of proteins with Coomassie blue. Figure 1A, lane 1 shows the protein profile of the treated seeds. Lane 2 relates to untreated control seeds. Lane 3 corresponds to the migration of standard proteins. Their molecular mass is indicated in kDa on the right of the photograph. The arrow indicates the migration of the SIP protein. We can see its strong induction during the pre-germination process of seeds.

(B) Treatment of beet seeds by osmo-conditioning in the presence of PEG 4aM1
The pre-germination experiments are conducted according to the osmoconditioning process. The beet seeds (one hundred seeds per batch) are incubated for 8 days at 200C in the presence of 6 ml of water containing PEG 6000

EXAMPLE 2. Revolution of the SIP protein during the germination of beet seeds
The germination experiments are carried out in Petri dishes (12 x 12 cm), in the dark (one hundred seeds and 6 ml of water per dish) and at a temperature of 20 "C. On the one hand, the revolution of the cumulative rate of germination as a function of time and on the other hand, samples are taken to produce crude extracts and quantify the rate of SIP protein as indicated.

FIG. 2A represents the evolution as a function of the incubation time at 200C of the cumulative rate of seed germination (o), and of induction of the SIP protein (9), the latter being revealed specifically by conducting ELISA tests with anti-SIP antibodies. The results concerning the SIP protein are expressed in arbitrary units (100% corresponding to the maximum rate obtained at 96 h of germination) and in relation to a seed. It can be seen that the SIP protein is present at a basal level (10 to 15% of the maximum level) in dry seeds and that its expression level is multiplied by a factor of 6 to 7 during germination. A significant amount of SIP is synthesized before the radicles appear. Finally, when the seed lot reaches its maximum germination percentage (between 90 and 130 h), the SIP protein slowly disappears
FIG. 2B represents the evolution of the protein profile in the protein extracts during imbibition and germination at 20 "C, after analysis of the proteins in 20% polyacrylamide gel containing SDS (PhastSystem from Pharmacia) and staining proteins with Coomassie blue Figure 2B, lanes 2, 3, 4, 5 and 6 show the protein profile of the control seeds, and of the seeds obtained after 13 h, 24 h, 36 h and 48 h of germination, respectively Lane 1 corresponds to the migration of standard proteins. Their molecular mass is indicated in kDa on the left of the photograph. The black arrow on the right indicates the migration of the SIP protein. Its strong induction can be seen during germination. at 20 "C.

EXAMPLE 3 Osmo-Qndlponnement seeds beet in the presence of polyvinyl glycol (PEG and induction of the protein SIP
The germination experiments are carried out at 200C and in the dark as described.

FIG. 3A represents the evolution of the cumulative rate of germination of the control seeds incubated in the presence of water alone (9), of the seeds incubated in the presence of PEG 6000 in water giving osmotic potentials of -3 bars (o) and from -7.5 bars (+). We can see the strong slowing of the root breakthrough for the samples incubated in the presence of
PEG 6000. This osmo-conditioning treatment leads on the one hand to the massive induction of the SIP protein and on the other hand to the pre-germination of beet seeds. FIG. 3B represents the evolution of the content of the seeds in protein SIP during the incubation for 200 h at 200C (no seed germinated, see FIG. 3A) in the presence of solutions of PEG 6000 giving osmotic potentials of - 7.5 bars, -10 bars, - 15 bars and -20 bars.

The results concerning the SIP protein are expressed in arbitrary units (100% corresponding to the maximum rate) and with respect to a seed. We can see the strong induction of the SIP protein during treatment. The seeds incubated at -7.5 bars, 20 "C and in the dark for 200 h are removed, washed quickly to get rid of the PEG, then dried to bring them to a hydration level close to that of non-control seeds. treated (10% water). These seeds are germinated at 20 "C, in the presence of water alone, as described. FIG. 3C represents the evolution of the cumulative germination rate of the seeds thus treated (O) and of the control seeds (9). It can be seen that the treated seeds have a strong lead to germination, attesting to the effectiveness of priming by osmo-conditioning.

EXAMPLE 4. Hydroconditioning of beet seeds in the presence of water at different temperatures and induction of the SIP protein
The experiments are carried out at different temperatures and in the dark as indicated. FIG. 4A represents the evolution of the cumulative rate of germination of seeds incubated at 200C (9), 100C (O) and 50C (+). We can see the great slowing down of the root breakthrough when the temperature of the germination experience decreases. This hydroconditioning treatment leads on the one hand to massive induction of the protein
SIP and on the other hand to the pre-germination of beet seeds. FIG. 4B represents the kinetics of induction of the SIP protein during the incubation at 50C (no seed germinated, see FIG. 4A). The results concerning the SIP protein are expressed in arbitrary units (100% corresponding to the maximum rate measured after two days of treatment) and in relation to a seed. We can see the strong induction of the SIP protein during treatment, compared to untreated control seeds. Seeds incubated at 50C and in the dark for 1 1 days are removed, then dried to bring them to a hydration level close to that of untreated control seeds (10%). These seeds are put to germinate at 15 "C, in the presence of water alone, as described. FIG. 4C represents the evolution of the cumulative germination rate of the seeds thus treated (O) and of the control seeds (9). find that the treated seeds have a strong germination advance, attesting to the effectiveness of this pre-germination method. Seed samples were also taken during the incubation at 50 C. All these samples were dried to bring them back to the hydration level of the untreated controls (10%), then put to germinate in the presence of water alone (6 ml per hundred seeds) at 150 C. FIG. 4D shows the evolution of the germination rate (expressed in % of seeds analyzed) measured after 63 h of incubation at 150C as a function of the level of SIP protein contained in the different samples taken during the first incubation at 5 "C. We can see the existence of a positive correlation between priming intensity and SIP content.

EXAMPLE 5. Dlstnbutlon of the SIP Protein in the Cycle of Beet Plants
The mature beet seeds (Universe variety) are put to germinate (T = 200C) at time zero either on filter paper in Petri dishes (100 seeds and 6 ml of water per box), or on potting soil to obtain plants. In the latter case, they are watered daily with water. Crude extracts of total and soluble proteins are prepared from various organs (dry seeds, seeds germinated after 96 h, cotyledons after 8 days of germination, roots and leaves after 48 days of germination). The results concerning the SIP protein are expressed in arbitrary units (100% corresponding to the maximum level measured in the germinated seeds after 96 h) and relative to one mg of total proteins in each of the extracts.

EXAMPLE 6 Presence of proteins equivalent to the SIP protein in other seed species
Different batches of seeds belonging to chenopodiaceae (beet, spinach), papilionaceae (lupine, bean, pea, alfalfa), compound (sunflower, chicory, matrix, lettuce), labiatae (lamier), cucurbits (zucchini, pickle), cruciferous plants (rapeseed , mustard, radish), cereals (barley, corn, wheat, rice), nightshade (tomato), umbelliferae (carrot) are put to germinate at 200C in Petri dishes as described for beet. When each batch reaches a germination percentage of 50%, the seeds are taken and crude protein extracts are produced as described for beet. These extracts are analyzed using an ELISA technique to quantify their level of reactivity towards anti antibodies.
Beetroot SIP. The results concerning the SIP protein are expressed in arbitrary units (100% corresponding to the level measured in beets) and relative to one mg of total protein in each extract. Figure 6A shows the existence of proteins recognizing anti-SIP antibodies in many species, with the exception of cereals. FIG. 6B shows that, for spinach chicory, lamier and zucchini, the level of proteins equivalent to SIP increases sharply during the early phases of germination.

EXAMPLE 7 Comparison of the molecular mass of the protein SIP in beetroot and other seed species
Different batches of seeds (sugar beet, spinach, chicory, louse, lupine, lamier) are put to germinate at 200C in Petri dishes as described for beet. When each batch reaches a germination percentage of 50%, the seeds are taken and crude protein extracts are produced as described for beet. These extracts are analyzed by electrophoresis in 20% polyacrylamide gel in the presence of SDS (PhastSystem from Pharmacia) and staining with Coomassie blue or revelation with anti-SlP antibodies after electrotransfer of the proteins of the gel onto a nitrocellulose sheet according to Towbin et al. (1979).

(A) Figure 7A shows the analysis of beet and spinach seeds after staining with Coomassie blue. Lane 2 corresponds to control beet seeds. Lane 3 corresponds to sprouted beet seeds. Lane 4 corresponds to control spinach seeds. Track S corresponds to germinated spinach seeds. It can be seen that, as with beet, sprouted spinach seeds contain a soluble protein mainly of about 22 kDa. The molecular mass of standard proteins is indicated in kDa (lane 1).

(B) FIG. 7B shows the same samples as in FIG. 7A, after electrophoretic transfer of the proteins onto the nitrocellulose sheet and reaction with anti-SIP antibodies. The numbering of the tracks is the same as in Figure 7A. The revelation is carried out with the secondary anti-rabbit antibody antibodies coupled to peroxidase (Sigma) and development of the peroxidase reaction.

(C) Figure 7C shows the analysis of germinated seeds of chicory (track 1), zucchini (track 2), spinach (track 3), lupine (track 4), lamier (track 5), matrix (track 6) and beet (lane 7) after electrophoresis, transfer of proteins to nitrocellulose sheet and reaction with anti-SIP antibodies. The revelation is carried out with the secondary anti-rabbit antibody antibodies coupled to peroxidase (Sigma) and development of the peroxidase reaction. The molecular mass of standard proteins is indicated in kDa. In the case of chicory, zucchini, spinach, anti-SIP antibodies reveal a small group of proteins (doublet or triplet), of molecular masses very close to that of the protein
Beetroot SIP. The synthesis of these proteins is strongly induced during germination.

 All of these results suggest a role for the SIP protein in the controlled water uptake of seeds during germination and during priming treatments (osmo-conditioning and hydro-conditioning). It should be noted that the SIP protein represents the majority soluble protein in soaked beet and spinach seeds. It could therefore be involved in the distribution of water molecules (and / or solutes) within the various constituent parts of the seed.

Within the framework of this hypothesis, it is interesting to compare the properties of the SIP protein with those of vegetable proteins recently described by the team of
Chrispeels in the United States [see for example Chrispeels & Maurel (1994) for a review].

These researchers have indeed demonstrated the existence in plants (beans,
Arabidopsis thaliana) membrane proteins linked to the tonoplast, which they called aquaponnes or TIP proteins fTonoplast lntrinsic Proteins'). It is a multigene family and some of these proteins are expressed specifically in seeds. They are found in particular in the membrane system surrounding the vacuoles where the seed reserve proteins are found, and their role would be to maintain the integrity of the tonoplast during the dehydration of the seeds at the end of maturation, and during their hydration during germination (Johnson et al., 1991). These aquaporins have molecular masses of the order of 27 kDa, very close to the mass of the SIP protein.

However, there are several indications that the two types of protein radically differ. a) The SIP protein is easily extractable in aqueous solvent, whereas the TIP proteins are extremely hydrophobic and can only be extracted from plant tissues in the pence of detergents (Triton for example). b) Seed-specific TIP proteins are abundant in mature seeds and disappear rapidly during gennination (Mader & Chrispeels, 1984; Johnson et al., 1989). In contrast, the SIP protein is at a basal level in mature seeds and is significantly induced during the early stages of germination.

 In conclusion, the SIP protein defines a new class of seed-specific proteins.

BIBLIOGRAPHICAL REFERENCES
Bewley, JD & Black, M. (1983) in Physiology and Biochemistry of Seeds in Relation to
Germination, Vol. 1, pp. 177-244, Springer-Verlag, Berlin
Chrispeels, MJ & Maurel, C. (1994) Plant Physiol. 105, 9-13
Coolbear, P., Newell, AJ & Bryant, JA (1987) Ann. Appl. Biol. 110, 185-194
Fincher, GB (1989) Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 305-346 lohnson, K D., Hennan, EM & Chrispeels, MJ (1989) Plant Physiol. 91, 1006
1013
Karsen, CM, Haigh, A., van der Toorn, P. & Weges, R. (1989) in Recent Advances in the Development and Germination of Seeds (Taylorson, RB, ed.) NATO ASI serins, Series A, Life sciences , flight. 187, pp. 269-280 Mader, M. & Chrispeels, MJ (1984) Planta 160, 330-340
Michel, BE & Kaufmann, MR (1973) Plant Physiol. 51, 914-916
Tarquis, AM & Bradford, KJ (1992) J. Exp. Bot. 43, 307-317 Teissère, M., Sergi, I., Job C. &! Job, D. (1990) Eur. J. Biochem. 193, 913-919
Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76.43504354.

The subject of the invention is also molecular, protein or nucleic probes, characterized in that they are derived from the protein (amino acid sequence of the protein, base sequence of the complementary DNA coding for the protein) above.

The present invention also relates to a process for obtaining transformed plants, characterized in that one inserts into the genome of plants which would not naturally express the SIP protein (or an equivalent protein), or which would express this protein but at a low level, one or more copies of the gene encoding the protein
SIP according to the invention, in order to express or overexpress this protein in seeds of plants transformed at the time of germination and to increase the germinative capacity of these seeds.

Claims (17)

1. Pure protein of plant origin, characterized in that it is obtained from seeds of a plant species, that it comprises at least one unit of 18 to 25 kDa, is expressed specifically in seeds, and appears rapidly on during the early stages of germination. 2. Protein according to claim 1, characterized in that the germination is carried out during a pre-germination process. 3. Protein according to one of claims 1 and 2, characterized in that it comes from a species of chenopodiaceae. 4. Protein according to claim 3, characterized in that it comes from the beet of spinach or lamb's quarters. 5. Protein according to one of claims 1 and 2, characterized in that it comes from a species of compounds. 6. Protein according to claim 5, characterized in that it comes from chicory or matrix. 7. Protein one of claims 1 and 2, characterized in that it comes from a species of labiatae. 8. Protein according to claim 7, characterized in that it comes from the lamia 9. Protein according to one of claims 1 and 2, characterized in that it comes from a species of cucurbits. 10. Protein according to claim 9, characterized in that it comes from zucchini. 11. Protein according to claims 1 to 10, characterized in that it is moreover specifically recognized by the antibodies directed against the beet protein SIP. 12. Proteins equivalent to the beetroot SIP protein according to claims 1  and 11, and obtained from different seeds according to claims 2 to 10. 13. Antibodies, called anti-SIP, characterized in that they recognize the protein  according to one of claims 1 to 11. 14. Molecular, protein or nucleic acid probes, characterized in that they are derived from the protein (amino acid sequence of the protein, base sequence of the complementary DNA coding for the protein) and anti-SIP antibodies (any polyclonal or monoclonal antibody specifically recognizing at least one epitope carried by the beet protein, according to one of claims 1 to 12. 15. Use of proteins capable of being induced during any process of imbibition of seeds, in particular during germination and during pre-germination processes, according to claims 1 to 12, or molecular probes according to claim 14 as a marker for the seed imbibition stage. 16. Device (kit) for determining the stage of imbibition of seeds, in particular during pre-germination processes (priming), characterized in that it is based on recognition by antibodies according to claim 13 which can be used in a technique ELISA. 17. Process for obtaining transformed plants, characterized in that plants which do not naturally express the SIP protein (or an equivalent protein) are inserted into the genome, or which would express this protein but at a low level, a or several copies of the gene coding for the SIP protein according to claims 1 and 10, in order to express or overexpress this protein in seeds of plants transformed at the time of germination and to increase the germinative capacity of these seeds.