EP2176660A2

EP2176660A2 - Use of the protein em6 as a performance marker for germination of seed lots and applications thereof

Info

Publication number: EP2176660A2
Application number: EP08831425A
Authority: EP
Inventors: Julia Buitink; Olivier Leprince
Original assignee: Agrocampus Ouest - Centre d'Angers; Institut National de la Recherche Agronomique INRA
Current assignee: Agrocampus Ouest - Centre d'Angers; Institut National de la Recherche Agronomique INRA
Priority date: 2007-07-17
Filing date: 2008-07-17
Publication date: 2010-04-21
Also published as: US20100285508A1; WO2009037390A2; WO2009037390A3

Abstract

The invention relates to the determination of the level, maintenance and/or performance of germination of a seed lot, by quantification of the protein EM6, belonging to the group of Late Embryogenesis Abundant (LEA) proteins. The present invention further relates to antibodies permitting the quantification of the protein EM6.

Description

USE OF THE EM6 PROTEIN AS A MARKER FOR THE GERMINATION PERFORMANCE OF LOTS OF SEEDS AND ITS APPLICATIONS.

The present invention relates to the use of the EM6 protein as a marker of the germination performance of seed lots.

One of the challenges of the seed industry is to obtain seed lot with high germination vigor, that is to say a good germination ability in a fast and uniform way but also a good quality of growth of the seeds. plants, regardless of the environmental and sowing conditions.

Several germination steps in non-dormant seeds have been identified. The phase I of imbibition is characterized by a passive influx of water which is made rapidly in the dry seed in latent life. Then a phase II reduction of the imbibition rate is followed by a last phase where again the seed absorbs water. It is during this last stage that there is an elongation of the radicle resulting in the germination, that is to say the protrusion of this one out of the seeds of the seed. During these three phases, the metabolism is gradually reactivated. On the other hand, during phases I and II of imbibition, the seed remains able to survive drying which brings its water content to values similar to those observed in an ungerminated seed. This property is referred to as desiccation tolerance. During drying, the cellular and metabolic events that lead to germination are stopped and the seed returns to a latent state of life. Tolerance to desiccation is lost progressively during the imbibition, first in the radicle when it has pierced the integuments and then in the other parts of the embryo.

In plants, the presence of LEA (Late Embryonic Abundant) proteins is correlated with desiccation tolerance. Notably, these proteins accumulate during seed maturation (Cuming, 1999, Kluwer Academy Press, The Netherlands, pp753-780) and they would have a role in protection against the effects of desiccation and other osmotic stress in both vegetative and seed tissues.

They are classified into 5 groups according to their sequence homology. More specifically, there is a correlation between the decrease in the amounts of certain LEAs of groups 1, 2, 3 and 5 and the loss of tolerance to desiccation during germination (Boudet et al, 2006, Plant Physiology, 140: 1418-1436 ). The EM6 protein belongs to the group 1 of the LEA proteins. Boudet et al (Plant Physiology, 2006, 140: 1418-1436) identified this protein in Medicago truncatula. The polypeptide sequence of this protein is accessible on Genbank under the number ABB13462 (Boudet et al Plant Physiology, 2006, 140: 1418-1436). They showed that it is particularly expressed in the radicles tolerant to desiccation, before their emergence. In addition, after germination, at the 2.8 mm protusion stage, the expression of this protein in the radicles become sensitive is increased by an osmotic treatment with polyethylene glycol (PEG) which restores the tolerance to desiccation.

EM6 homologues have been identified in many other higher plants: by way of non-limiting examples, mention may in particular be made of Arabidopsis thaliana (Genbank AAD25932, NP181546, Swissprot Q02973, Q07187), Glycine max (Swissprot P93165), Vigna radiata ( Genbank AAB07225), Oryza sativa (Swissprot P46520), Triticum aestivum (Swissprot Q9ZR71), Hordeum vulgare (Swissprot P46532), Dry cereal (Swissprot Q9M4P1), Zea mays (Swissprot P46517), Raphanus sativus (Swissprot P11573), Brassica napus (Swissprot 065 725),

Lycopersicum esculentum (TAIR Gene Index TC178231),

Phaseolus aureus (Swissprot Q41685), Vitis vinifera (TrEMBL

A5BF16), Daucus carota (Swissprot Q5KTS6), Helianthus annuus

(Swissprot P46514). Many seed treatment processes

(pregermination, priming), aimed at activating seed germination to improve germinative pre-market performance are well known. The seeds thus treated are more vigorous, germinate and / or raise faster or better in penalizing conditions. It is essentially to produce pregerminated seeds, ie seeds in which the radicle has not yet emerged, by means of a controlled treatment of imbibition by hydration, followed by drying. The imbibition can take place with an osmoticum such as PEG or salts (KNO3) or without osmoticum. The seeds are then dried which reduces their water content to values similar to those observed for ungerminated seeds.

Some pregermination methods are based on the treatment of seeds at more advanced stages, after the emergence of the radicle (EP 0 202 879). However, at this stage the seed is much more fragile. There are processes implementing a hydration step and the addition of a gel (Agrigel) in water or an encapsulating polymer (US 4,780,987).

In addition to the pregerminative treatments, post-harvest packaging methods may be applied to the seeds including imbibition steps. These are coating processes, film coating or treatment with chemicals. These conditioning methods can also be applied independently of the pregerminative treatment. The pregerminative treatment must be carried out under conditions that allow the germination to be initiated in a homogeneous manner and that it is stopped early enough for the seed to be able to reinitiate the germination effectively. There is therefore a need to accurately determine the conditions of the imbibition treatment, including the duration of the treatment, to meet the seed quality criteria. If it is too short, no beneficial effects on germinative performance are observed. At worst, there is no initiation of germination. Overly long duration of the seed imbibition treatment has the effect of reducing the longevity and germinative performance of the dried treated seeds, which makes their conservation and management of seed stocks more problematic, if not impossible.

In addition, if the treatment is poorly conducted and fails (because of the very heterogeneous nature of the seed lots or due to a handling error), the lot is lost. It is therefore important to be able to follow the imbibition treatment so that it can be adapted if necessary during the process.

The physiological quality of the seed lot can be defined in particular in terms of germination performance and / or storage ability as well as level of heterogeneity of the batch.

The germination performance (or T (50)) of a seed lot is estimated for example by the germination rate expressed in hours to reach 50% germination after sowing under specific conditions defined for each species or variety according to the standards. ISTA (International Seed Testing

Association). There are other methods published in the literature such as measures of germination under water stress (Bettey and Finch-Savage, 1996, Seed

Science Research, 6: 165-173)

The storage ability (or P50) is defined by the aging time to obtain 50% germination for a batch of seeds at a precise and controlled relative humidity and temperature, for example 75% relative humidity and

35 ° C. This conservation suitability test is known to seed companies as an indicator of germination performance (Ibrahim et al, 1993, Journal of Seed

17: 29-37 and Ellis and Roberts, 1981, Seed Science and Technology, 9: 373-409).

However, the time required to carry out the tests indicated above is from several days to several weeks and is often longer than the pregermination treatment itself which is 3 to 7 days.

In order to circumvent this problem, molecular markers allowing the monitoring of pregermination have been characterized. However, the method of using the The label remains heavy to use (by measurements of enzymatic activity or of soluble sugar content,

(Bettey and Finch-Savage, 1996, Seed Science Research, 6: 165-

173; EP 0 202 879) or the marker itself is not specific to the seed.

The inventors have found, during the imbibition of seeds of Medicago truncatula, a significant linear correlation between, on the one hand, the content of EM6, measured using an antibody directed against said EM6 protein, in a soluble protein extract. non-emerged radicle and, secondly, the ability to preserve the seeds concerned. They further observed that the coefficient of linearity between the amount of EM6 and the storage ability remains stable even when different imbibition conditions are applied to the seeds.

The inventors have furthermore found that the antibody directed against EM6 of Medicago truncatula recognizes the homologous proteins in seeds or embryos of other dicotyledonous and monocotyledonous species, and that the linear correlation between the amount of protein recognized by this antibody in the non-emergent radicle, and the ability to conserve is also observed in these other species.

It thus appears that the EM6 protein is a robust marker and perfectly correlated with the ability to preserve, and thus allowing to follow the evolution of it during the imbibition treatments of a batch of seeds.

The inventors have furthermore identified, from the Medicago truncatula EM6 protein, two peptides making it possible to obtain antibodies capable of recognizing homologues of EMβ in other plant species, and that can be used as the antibody directed against the protein. Whole EM6, to quantify this protein, and to follow its evolution during an imbibition treatment.

The present invention relates to a method for determining the storage ability of a seed lot undergoing an imbibition treatment, characterized in that it comprises the quantification in a seed sample taken from said batch of proteins recognized by an antibody chosen from: a) an antibody recognizing a protein, hereinafter referred to as EM6 protein, and having at least 70%, preferably at least 75% and most preferably at least 80% identity, or at least 80%, preferably at least 85% and most preferably at least 90% similarity with the EM6 protein of Medicago truncatula identified by accession number GenBank ABB13462 (and also represented in the attached sequence listing under the number SEQ ID NO: 1), and containing a region defined by the general sequence (I) GX _I SX ₂ GGX ₃ TRX ₂ X ₄ QX _S G (SEQ ID NO: 2) wherein X is H or R, X2 is K or R, X 3 is Q or N, X ₄ is E or D, X ₅ is L or M, or by the general sequence (I bis) GXiSX ₂ X ₃ GX ₄ XsQXeG TRX ₂ (SEQ ID NO: 6) wherein X represents feel H or R, X ₂ is H, K or R, X ₃ is G or A, X ₄ is Q, H, S or N, X ₅ is E, Q or D, X ^ represents L, I or M, and a region defined by the general sequence (II) XIQX ₂ X ₃ X ₄ XSGYX ₆ X ₅ MGX ₇ X ₈ (SEQ ID NO: 3) where X ₁ represents D or E, X ₂ represents L, or M, X ₃ is G or S, X ₄ is T, E or Q, X ₅ is E or Q, Xe represents K, H, Q, S or R, X ₇ is R or K and X is K or Q, or by the sequence general (IIa) X ₁ QX ₂ X ₃ X ₄ XsGYX ₆ X ₅ MX ₇ X ₈ X ₉ (SEQ ID NO: 7) where X _x is D, Q or E, X ₂ is L, I or M, X ₃ is G or A, X ₄ is S, T, G, E, H, K or Q, X ₅ is E or Q, X ₆ is K, H, Q, S or R, X ₇ is G or A, X ₈ is R, K, P or H and Xg is K or Q; b) an antibody directed against a portion of said EM6 protein comprising at least one of sequences (I), (Ia), (II) or (IIa).

According to a preferred embodiment of the method of the present invention, an antibody directed against a portion of said EM6 protein comprising at least one of the sequences GRSKGGQTRKEQLG (SEQ ID NO: 4) or EQLGTEGYQEMGRK (SEQ ID NO: 5). Unless otherwise specified, the percentages of identity and similarity referred to herein are calculated using BLAST software (Altschul et al,

1997, Nucleic Acids Res., 25: 3389-402), with the default settings.

The process according to the invention can be carried out during an imbibition stage which can take place during a post-harvest conditioning and / or a pre-germinating treatment or any other treatment resulting in a modification of the water content of the seed or part of the seed either in time or in its distribution within the tissues of the seed.

By imbibition treatment is meant any treatment containing at least one step of hydration of the seeds, which can be carried out either in the presence of an inert osmoticum such as PEG or a salt (for example KNO3) or without osmoticum (for example , hydroconditioning, soaking). Among the imbibition treatments there may be mentioned pregermination treatment, and various post-harvest packaging methods of the seed such as coating or film coating. Hydration methods can be varied and include, for example, the use of water vapor, contacting the seeds with a liquid film or soaking the seeds. The graphical representation of the correlation between the quantity of EM6 detected by the process according to the invention and the storage ability and / or the germinative performance can be obtained from at least 3 points, each corresponding to one batch of seeds having undergone imbibition treatments of different durations. The coefficient of linearity can be directly deduced, which makes it possible to determine quantitatively the storage ability, irrespective of the imbibition treatment subsequently applied. Those skilled in the art may, by quantifying the content of EM6 protein of the sample, for example compare the effects of different operating conditions of a given treatment on the ability to preserve or performance germinative. He may also compare the effects of an imbibition treatment over time.

The process according to the invention may advantageously be carried out during the pregermination treatment in order to determine the duration of the treatment to obtain a seed lot with a desired preservation ability. Since the amounts of EM6 protein evolve linearly with the storage ability, the observed variations in amounts of EM6 protein are equal to the variations in storage ability.

Throughout the processing of the seed samples are taken to determine the content of EM6. The treatment is stopped for a content of EM6 corresponding to the desired storage ability value. For example, for older seed lots, a short imbibition leads to an increase in EM6 content.

If a person skilled in the art does not know the value of the coefficient of linearity of the species or of the variety or of the batch of seeds, he may, prior to the treatment itself, determine on a sample of seeds to be treated the value of the coefficient. linearity as defined above.

The process according to the invention can be carried out on whole seeds, or on embryos or non-emerged radicles previously isolated from the seeds.

Advantageously, the method according to the invention can be implemented separately on individual seeds taken from the batch to be tested, this makes it possible to determine the level of heterogeneity of a seed lot for the ability to preserve.

The subject of the present invention is also any anti-EM6 antibody directed against a region of said EM6 protein chosen from the region defined by the sequences

(I) or (I bis) and the region defined by sequences (II) or (II bis) above.

An antibody against a region of an EM6 protein is defined as any antibody capable of detectably binding to the region concerned on the protein. Whole EM6 or a fragment thereof, but not detectably binding to another region of the same protein. In the case of monoclonal antibodies, this includes any antibody recognizing an epitope located in said region; in the case of polyclonal antibodies, this includes any antibody preparation that does not show significant cross-reactions with other regions of the EM6 protein.

On the other hand, this antibody will advantageously have cross-reactions with the homologous region of EM6 proteins of other plant species. In particular, as indicated above, antibodies directed against the region defined by the sequence (I) or by the sequence (Ia) generally have a very broad specificity and can recognize a wide variety of EM6 proteins in monocotyledons or dicotyledons. , while the antibodies directed against the region defined by the sequence (II) or by the sequence (II bis) generally have a more restricted specificity. Antibodies according to the invention can be obtained by methods well known per se. Conventionally, an EM-6 protein fragment (in the form of a natural, recombinant, or synthetic peptide) comprising the region against which it is desired to direct the antibody, or at least one fragment of this region of size, is used as immunogen. sufficient to constitute a B epitope (usually at least 5 to 7 amino acids). If necessary, said peptide is mixed with an adjuvant, or coupled to a carrier protein, in order to increase its immunogenicity. The resulting antibodies can then be purified, also known per se. Generally, this purification comprises at least one affinity chromatography step on a column on which is grafted the peptide against which the antibody is to be directed. The entire EM-6 protein can also be used as an immunogen, and affinity chromatography can be performed on a column onto which the peptide to which the antibody is to be directed is grafted. By way of nonlimiting examples of anti-EM6 antibodies directed against a region defined by the general sequence (I), mention may be made of antibodies directed against the peptide defined by the sequence GRSKGGQTRKEQLG (SEQ ID NO: 4). As non-limiting examples of anti-

EM6 directed against a region defined by the general sequence (II), there may be mentioned antibodies directed against the peptide defined by the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5).

The present invention also relates to a

LO method of quantifying EMβ in plant material, characterized in that it comprises bringing said plant material into contact with an anti-EM6 antibody according to the invention, as defined above.

The present invention also relates to

The use of an anti-EM6 antibody according to the invention for determining the storage ability of a seed lot.

The present invention will be better understood with the aid of the additional description which follows, which refers to

2.0 examples illustrating the demonstration of the linear correlation between the amount of EMβ and the ability to preserve.

EXAMPLE I: CORRELATION BETWEEN CONSERVATION ABILITY AND THE QUANTITY OF EM6 DETERMINED IN ELISA AT MEDICAGO TRUNCATULA.

Treatment: imbibition and priming.

A batch of seeds of Medicago truncatula (var Parragio) is divided into 100 seeds which are soaked in a 9 cm petri dish or on Whatman No. 1 filter paper soaked in 4 ml of distilled water or on two Whatman No. 1 filter papers soaked with 7 ml of a solution of polyethylene glycol (Sigma, Mw 6000-8000) equivalent to water potentials calculated from the formula of Michel and Kaufmann, 1973 (Plant Physiology 51: 5 914-916). The petri dishes are then placed in the dark at 20 ^° C. for different times. Then, the seeds are optionally rinsed quickly with running water to remove the PEG. They are quickly dried in an enclosure ventilated at 43% relative humidity at 20 ⁰ C in the dark for 3 days. Aging treatment

The untreated or previously hydrated seeds then dried are placed on a grid in a hermetically sealed box containing a relative humidity of 75%. This is obtained by depositing at the bottom of the box a saturated solution of NaCl. The seeds are thus incubated at 35 ° C in the dark for increasing times. Over time, 100 seeds are removed from the can and sprouted in distilled water as described above. After 7 days, count the number of sprouts. A seed is considered sprouted when the radicle has emerged out of the seeds of the seed. The storage ability is expressed in days required to obtain 50% germination for each batch of aged P seeds (50). Protein extraction and ELISA

The protocol for preparing the protein extracts from the radicles of Medicago truncatula and that of the ELISA tests are described in Job et al., Seed Science Research, 1997-225-243. The proteins present in the soluble extracts are then assayed according to the Bradford method (using the Biorad Protein Assay kit, Biorad). The following biological samples were prepared using this method:

- Radicles isolated from whole seeds imbibed without osmoticum respectively during 3, 7, 11 and 15 hours at 20 ⁰ C in the dark. isolated radicles of whole seeds impregnated for 1 day and 3 days in an aqueous solution of PEG at a water potential of -IMPa and at -1.5 MPa at 20 ^° C. in the dark.

Sample preparation can be done in a standard 96-well ELISA microtiter plate (Greiner Bio-One ref 655161). The soluble protein extracts obtained by the method described above are diluted in PBS buffer (15OmM NaCl, 10mM Na ₂ HPO ₄ ) at 100 ng / .mu.l. 2μl of these Dilutions are added to 248 μl of PBS buffer. Then, successive half dilutions are prepared from the most concentrated extract taking care to homogenize the mixtures well. A volume of 100 μl of each concentration for each sample is transferred to an ELISA microtiter plate. The plate with the samples is incubated for 30 minutes at 35 ° C., then washed 5 times with 125 μl of

(PBS, 0.05% Tween-20). The rabies anti-EM6 primary antibodies directed against the entire recombinant EM6 protein of Medicago truncatula, or anti-EM6-peptide II directed against the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5) of the EM6 protein of Medicago truncatula are diluted to 1 / 1000 in PBS buffer. 100 μl of antibody solution are deposited per well. The plate is incubated for 30 minutes at 35 ° C. and then washed 5 times with 125 μl of buffer (PBS + 0.05% Tween-20). The secondary antibodies coupled to peroxidase (Anti Rabbit IGG Alkaline Phosphatase, Sigma) are diluted to 1/10 000 in PBS alone. 100 μl of antibody solution are deposited per well. The plate is then incubated for 30 minutes at 35 ° C. and then washed 5 times with 125 μl of (PBS + 0.05% Tween-20).

The microplate is revealed by the addition of a solution containing the aforementioned peroxidase substrates (ABTS, Sigma). 100 μl of ABTS developer are deposited per well. The reaction is monitored spectrophotometrically at 405 nm in a microplate reader, the intensity of the staining being proportional to the amount of antigen in the soluble protein extract. The absorbance is measured every 5 to 10 minutes and this during Ih. To determine the value of the relative content of EM6, the slope of the regression line of the absorbance values is calculated as a function of increasing concentrations of protein extracts.

The results are illustrated in Figures 1 and 2.

Legend of Figure 1:

A:% germination at 20 ° C as a function of aging time during storage at 35 ° C, 75% RH following an imbibition treatment in distilled water of increasing duration. The control corresponds to non-soaked seeds.

B:% germination at 20 ^° C. as a function of the aging time during storage at 35 ° C., 75% RH following an imbibition treatment in the presence of osmoticum

(polyethylene glycol) corresponding to two water potentials (-1 MPa and -1.5 MPa) for 1 or 3 days. The control corresponds to non-soaked seeds. Legend of Figure 2:

Correlation between the amount of EM6 and the longevity ability P (50) on batches of Medicago truncatula seeds imbibed in distilled water in the presence of osmoticum (PEG) (circles) and without osmoticum (triangles). The modalities of the imbibition treatments are (3, 7, 11 and 15 hours of imbibition in water, 1 or 3 days in a solution of PEG corresponding to a water potential of -1.5 or -1.0 MPa ). The amount in EM6 is determined in extracts of soluble proteins by ELISA radicles with one hand an anti-EM6 rabbit antibody directed against the whole recombinant protein EM6 Medicago truncatula (A) and secondly the ^λ antibodies -EM6-peptide II directed against the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5) of the Medicago truncatula EM6 protein (B) The control represents ungerminated seeds. The

Figure 1 shows that regardless of the duration of the imbibition, the percentage of germination measured after 7 days of imbibition decreases as a function of the duration of aging. Figure 1 determines the storage ability (P50) for each treatment. It is found that the longer the duration of the imbibition treatment increases the lower the value of P (50), regardless of the type of imbibition treatment applied (in distilled water (A) or in an aqueous solution of PEG (B)). Figure 2 shows that there is a significant linear relationship between the content of EM6, determined by an ELISA test with two different antibodies and P (50) obtained according to the modalities of the imbibition treatment. Moreover, the correlation coefficient is conserved whatever the type of imbibition treatment used (with or without osmoticum). The ELISA test put in place makes it possible to distinguish seeds of Medicago truncatula pretreated according to their aptitude for conservation.

EXAMPLE II: CORRELATION BETWEEN GERMINATION PERFORMANCE AND THE QUANTITY OF EM6 AT MEDICAGO TRUNCATULA.

Protein samples extracted from radicles isolated from treated seeds are prepared according to the protocol described in Example 1. To determine the rates of imbibition (T50), the treated seeds are dried as described in Example 1. Next 100 seeds are imbibed at 20 ⁰ C as described in Example 1. The sprouted seeds are counted every 2 hours. The germination percentages are plotted against the imbibition time to determine the time required to obtain 50% germination for each seed lot (T20).

The results are summarized in Figure 3. Legend of Figure 3:

Correlation between the amount of EMβ and germination performance, measured by the rate of T (50) germination on batches of Medicago truncatula seeds imbibed in distilled water in the presence of osmoticum (PEG) (triangles) and without osmoticum (circles). The modalities of the imbibition treatments are: 3, 7, 11 and 15h hours of imbibition in water; 1 or 3 days in a PEG solution corresponding to a water potential of -1.5 or -1.0 MPa). The quantity of EM6 is determined in extracts of soluble radicle proteins by ELISA test with on the one hand an anti-rabbit anti-EM6 antibody directed against the whole recombinant EM6 protein of Medicago truncatula (A) and on the other hand the antibody anti-EM6-peptide II directed against the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5) of the Medicago truncatula EM6 protein (B)

Beforehand, it has been verified that the longer the duration of the imbibition treatment, the lower the T (50). The results in Figure 3A and B show that there is significant linear correlation between T50 and EM6 content determined by ELISA in the presence of two different antibodies. Thus the content of Em6 is closely correlated with the germination vigor of the batches. EXAMPLE III REACTIVITY OF ANTI-EM6 ANTIBODIES ON PROTEIN EXTRACTS OF DICOTYLEDONES AND MONOCOTYLEDONES

Further experiments were performed on seed lots from other species to determine whether anti-EM6-peptide I and anti-EM6-peptide II antibodies recognized EMβ homologs in these other species.

The results are summarized in Figure 4.

Legend of Figure 4:

A + C: Antigen-antibody specific recognition by the Western blot technique using soluble protein extracts of seeds of 13 species (lane 1 to 13) using the anti-EM6-peptide II antibody directed against the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5) Medicago truncatula EM6 protein. Beforehand, 2.5-30 μg of proteins according to the species were separated by gel electrophoresis according to their molecular weight (molecular marker indicated on the left corridor) and transferred to nitrocellulose membrane. The assignment of the corridors to each species and the corresponding protein concentration are as follows: 1, celery (25 μg of protein); 2, carrot (25μg); 3, cucumber (30 μg); 4, leek (15 μg); 5, chives (15 μg); 6, onion (15μg); 7, impatience (15 μg); 8, petunia (15 μg); 9, thought (15 μg); 10, radish (2.5 μg); 11, broccoli (2.5 μg); 12, rapeseed (2.5 μg); 13 cabbages (2.5 μg). B + D: Antigen-antibody specific recognition by Western blot technique using soluble protein extracts of 13 species seeds (lane 1 to 13) using the anti-EM6-peptide I antibody directed against the sequence GRSKGGQTRKEQLG (SEQ ID NO: 4) Medicago truncatula EM6 protein. The assignment of the corridors and protein concentrations for each species are identical to those mentioned above. E + F: Antigen-antibody specific recognition by Western blot technique from soluble protein extracts of radicles isolated from 7 species using anti-EM6-peptide II (E) and anti-EM6-peptide I (F) The assignment of corridors to each species and the corresponding protein concentration are as follows: 14, Medicago truncatula (30 μg); 15, cowpea (15 μg); 16, soy (15 μg); 17, pea (15 μg); 18, beans (15 μg); 19, but (15 μg ⁾ • The protein samples extracted from dry whole seeds or radicles isolated from dry seeds are prepared according to the protocol described in Example 1. After electrophoresis on polyacrylamide gel 12% (m / v), the The gels are equilibrated twice 10 min in transfer buffer (8 mM Tris HCl [pH 6.8], 61 mM glycine, 20% (v / v) methanol).

Polyvinylidine difluoride (PVDF) or Immobilon membranes

(Millipore) are incubated for 10 seconds in pure methanol and then equilibrated in the transfer buffer. The semi-dry transfer (Biorad) is carried out under a current of 15V for 15 minutes. Detection is performed according to Boudet et al, 2006, Plant Physiology, 140: 1418-1436. The dilution of the primary anti-EM6-peptide I and peptide II antibody is 1 / 10,000th. The revelation is carried out by chemiluminescence using the kit Immun-Star Western C (Biorad), under the conditions recommended by the manufacturer. Shooting is carried out with the Chemidoc imager (Biorad) after optimization of the signal exposure according to the conditions recommended by the manufacturer.

These results show that there is a cross-reaction between the antibodies directed against the peptide sequences of the Medicago truncatula EM6 protein and the EM6 homologues of dicotyledons or monocotyledons. For most species, one or two major bands are detected by both antibodies. These results demonstrate the specificity of the antibody and the existence of several common epitopes in a significant amount. EXAMPLE IV: CORRELATION BETWEEN SUNFLOWER SEED CONSERVATION FITNESS AND EM6 QUANTITY DETERMINED BY ELISA

Experiments were carried out to validate on the sunflower the correlation between the abundance of EM6 and the germinative performance after imbibition treatments with or without osmoticum (PEG).

Imbibition treatment and priming

A batch of sunflower seeds is divided into units of 80 seeds which are imbibed in the presence of distilled water or an aqueous solution of PEG as described in Example I. The seeds are then placed in the dark at 20 ° ^C. C for increasing periods of time (Oh, 6h and 15h in water and 48h in PEG solution at -1.5 MPa). Then, they are optionally quickly rinsed with running water to remove the PEG and then quickly dried in a ventilated enclosure at 43% relative humidity at 20 ⁰ C in the dark for 3 days. Aging treatment Untreated or previously hydrated seeds then dried are aged according to the protocol described in Example I. During storage, 80 seeds are removed successively and germinated under the conditions described in Example I. After 7 days, count the number of sprouts. Storage ability is measured by P (75), defined by the aging time required to obtain 75% germination for the seed lot.

Protein extraction and ELISA test The protocol for preparing protein extracts from isolated radicles and the quantification of the proteins are described in Example I. The preparation of the samples for the ELISA test is described in Example I except that the dilutions successive half are made from a protein concentration of 100 ng / μl. The procedure described in Example I was followed using the anti-EM6-peptide II antibody directed against the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5) of the EM6 protein of Medicago truncatula.

The results are illustrated in Figure 5.

Legend of Figure 5: Relation between the relative amount of EM6

(determined by ELISA from soluble protein extracts of radicles isolated using the anti-EM6-peptide II antibody) and the longevity ability of sunflower seeds having undergone different imbibition treatments. The seeds were soaked for increasing times (shown in the figure) in distilled water (circles) or 48 h in the presence of an aqueous solution of PEG corresponding to a water potential of -1.5 MPa (circles). The longevity ability here corresponds to the time required to obtain 75% germination after aging after storage at 35 ^° C., 75% RH.

Used in the ELISA tests as described for Medicago truncatula, the anti-EM6-peptide II antibody gives in sunflower the same result as in the examples described above. In particular, Figure 5 shows that there is a linear correlation relationship (correlation coefficient = 0.971) between the relative amount of EM6 and the longevity P (75) obtained for the different imbibition treatments. The longer the longevity decreases, the more the relative content of EM6 decreases proportionally regardless of the type of imbibition treatment used. Moreover, these results also indicate that it is not imperative to carry out an aging experiment sufficiently long to reach the value of P50. EXAMPLE V: CORRELATION BETWEEN THE AMOUNT IN EM6 DETERMINED BY ELISA AND IMPROVING THE ABILITY TO PRESERVE A LOT OF AGEED OIL SEED BY SOAKING IN DISTILLED WATER.

One of the objectives of a pregerminative imbibition is to eliminate the effects of aging suffered by a batch of seeds too old or stored in bad conditions. Experiments have been carried out to show that EMβ is correlated with longevity ability as it evolves depending on the duration of soaking in aged onion seeds.

Treatment: d / imbibition and aging

A lot of naturally aged onion seeds is divided into units of 80 seeds and imbibed according to the protocol described in Example I. Then, the seeds are quickly dried in a ventilated enclosure at 43% relative humidity at 20 ^° C. in the dark for 3 days. The procedure described in Example I is followed for the seed aging treatment.

Protein extraction and ELISA test

The protocol for the preparation of the protein extracts and the quantification of the proteins are described in Example I. The preparation of the samples and the ELISA test are described in Example I except that the successive one-half dilutions are made from a protein concentration of 1 μg / μl. In this experiment, the anti-EM6-peptide II antibody directed against the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5) of the Medicago truncatula EM6 protein was used.

The results are shown in Figure 6.

Legend of Figure 6:

A. Effect of the duration of the imbibition in distilled water at 20 ⁰ C in the dark of naturally aged onion seeds on the ability to longevity (P (50).

B. Relation between the relative amount of EM6 determined by ELISA using the anti-EM6-peptide II antibody directed against the sequence EQLGTEGYQEMGRK (SEQ ID NO: 5) of the Medicago truncatula EM6 protein and the longevity P (50) according to increasing imbibition times

(shown in the figure).

Figure 6A shows that between 10 and 17 hours of imbibition, the P50 value increases from 8 to 12.5 days.

Then, it decreases after 24 hours of imbibition and stabilizes around a value of P50 equivalent to 9-10 days. In the

FIG. 6B shows the content of EM6 as a function of the set of P50 values obtained after a period of time. croissants of imbibition. It is observed that there is a linear relationship between the relative amount of EM6 in the radicles and the longevity ability.

EXAMPLE VI: CORRELATION BETWEEN THE QUANTITY IN EM6 DETERMINED BY WESTERN BLOT AND IMAGE ANALYSIS AND IMPROVING THE ABILITY TO PRESERVE A LOT OF BROCOLI SEEDS.

It is not always possible (due to lack of time or a sufficient quantity of seeds) to carry out an exhaustive study of the loss of the germinal faculty during aging, as described in example 1. The experiment below shows that the content of EM6 predicts the longevity of broccoli seeds when only one point of aging is achieved. It also shows that the determination of EM6 can be made by Western blot combined with quantification of the signal by image analysis. Treatment: of imbibition and aging

A batch of broccoli seeds is divided into a unit of 100 seeds and imbibed according to the protocol described in Example I. The Petri dishes are then placed in the dark at 20 ⁰ C for increasing periods of time (6h and 15h in water and 24 and 48h in the PEG solution corresponding to a water potential of -1.5 MPa, then the seeds are optionally rinsed with running water in order to remove the PEG and are rapidly dried in a chamber ventilated at 43% relative humidity at 20 ^° C. in the dark for 3 days The untreated or previously hydrated seeds then dried are then stored in relative humidity at 35 ° C. according to the protocol described in Example I. After 19 and 25 days of aging, 100 seeds are germinated under the conditions described in Example I. After 7 days, the number of sprouts is counted.

Protein extraction, Western blot and signal quantization

The protocol for the preparation of protein extracts from isolated radicles and the quantification of proteins are described in Example I. One μg of protein extracts is separated by gel electrophoresis. polyacrylamide 12% (w / v) as described in Example II and transferred to polyvinylidine difluoride immobilon membrane

(Mill-ipore). The semi-dry transfer (Biorad) is carried out under a current of 15V for 15 minutes. The detection of EM6 is carried out according to Boudet et al, 2006, Plant Physiology, 140:

1418-1436. The dilution of the primary anti-EM6-peptide I antibody directed against the sequence GRSKGGQTRKEQLG (SEQ ID NO:

4) Medicago truncatula EM6 protein is 1 / 10,000th. The revelation is carried out by chemiluminescence using the kit Immun-Star Western C (Biorad) under the conditions recommended by the manufacturer. The shooting and the quantification of the signal are carried out by means of an imager (Chemidoc Biorad) after optimization of the exposure according to the conditions recommended by the manufacturer. The results are shown in Figure 7.

Legend of Figure 7:

Relation between relative amount of EM6 in isolated radicules of broccoli seeds undergoing different imbibition treatments and percentages of germination obtained after 19 (triangle) and 25 (circle) days of aging at 35 ° C, 75% RH. Each symbol represents different imbibition modalities which are indicated in the Figure. EM6 was quantified by Western blot analysis using the anti-EM6-peptide I antibody. The longevity of seeds undergoing different imbibition treatments was measured after 19 (triangles) and 25 minutes. (circles) aging days at 75% RH and 35 ° C. Figure 7 shows that there is a linear relationship (correlation coefficient = 0.92 and 0.97) between the longevity and the relative amount of EM6 in broccoli radicles: plus germination performance measured by a single point of aging. decreases and the relative content of EM6 decreases proportionally regardless of the type of imbibition treatment used. The results also show that the Western blot technique and image analysis can advantageously replace the ELISA test.

Claims

1) A method for determining the storage ability of a seed lot undergoing an imbibition treatment, characterized in that it comprises the quantification in a seed sample taken from said batch of proteins recognized by an antibody selected from: a) an antibody recognizing a protein, hereinafter referred to as EM6 protein, and having at least 70%, preferably at least 75% and most preferably at least 80% identity, or at least 80% identity; %, preferably at least 85% and most preferably at least 90% similarity to the Medicago truncatula GenBank ABB13462 EM6 protein (SEQ ID NO: 1), and containing a region defined by the general sequence (I ) GX ₁ SX ₂ GGX ₃ TRX ₂ X ₄ QX ₅ G (SEQ ID NO: 2) wherein X is H or R, X ₂ is K or R, X ₃ is Q or N, X ₄ is E or D, X ₅ represents L or M, or by the general sequence (I bis) GXiSX ₂ GX ₃ X ₄ TRX ₂ X ₅ QXeG (S EQ ID NO: 6) wherein X _x is H or R, X ₂ is H, K or R, X ₃ is G or A, X ₄ is Q, H, S or N, X ₅ is E, Q or D, X ₆ represents L, I or M, and a region defined by the general sequence (II) XIQX ₂ X ₃ X ₄ X ₅ GYX ₆ X ₅ MGX ₇ X ₈ (SEQ ID NO: 3) where X is D or E, X ₂ is L or F, X ₃ is G or S, X ₄ is T, E or Q, X ₅ is E or Q, X ₆ is K, H, Q, S or R, X ₇ is R or K and Xa represents K or Q, or by the general sequence (IIa) XiQX ₂ X ₃ X ₄ X ₅ GYX ₆ X ₅ MX ₇ X ₈ X ₉ (SEQ ID NO: 7) where X is D, Q or E, X ₂ is L, I or M, X ₃ is G or A, X ₄ is S, T, G, E, H, K or Q, X ₅ is E or Q, X ₆ is K, H, Q, S or R, X ₇ is G or A, X ₈ is R, K, P or H and Xg is K or Q; b) an antibody directed against a portion of said EM6 protein comprising at least one of sequences (I), (Ia), (II) or (IIa). 2) Process according to claim 1, characterized in that an anti-EM6 antibody chosen from: an antibody directed against a peptide of sequence GRSKGGQTRKEQLG (SEQ ID NO: 4)

an antibody directed against a peptide of sequence EQLGTEGYQEMGRK (SEQ ID NO: 5).

3) Anti-EM6 antibody selected from:

an antibody directed against a peptide defined by the general sequence SEQ ID NO: 2;

an antibody directed against a peptide defined by the general sequence SEQ ID NO: 6;

LO - an antibody directed to a peptide defined by the general sequence SEQ ID NO: 3;

an antibody directed against a peptide defined by the general sequence SEQ ID NO: 7.

4) anti-EM6 antibody according to claim 3, L5 selected from:

an antibody directed against the peptide of sequence SEQ ID NO: 4;

an antibody directed against the peptide of general sequence SEQ ID NO: 5.

5) Method for quantifying EM6 in plant material, characterized in that it comprises bringing said plant material into contact with an anti-EM6 antibody according to any one of claims 3 or 4.

6) Use of an anti-EM6 antibody according to any of claims 3 or 4 to evaluate the storage ability of a seed lot.