Seed quality and germination

1 downloads 0 Views 3MB Size Report
Oct 14, 2006 - gel electrophoresis (2D-PAGE) as described earlier (Görg et al., 1987; Rajjou et al.,. 2004). ... 2D Elite version 4.01 software (Amersham Biosciences). .... et al., 2001, 2002a; Rajjou et al., 2004; http://seed.proteome.free.fr).
34

Seed Quality and Germination

L. RAJJOU,1 Y. LOVIGNY,1 C. JOB,1 M. BELGHAZI,2 S. GROOT3 AND D. JOB1 1Centre

National de la Recherche Scientifique (CNRS)-Bayer CropScience Joint Laboratory, (UMR CNRS 2847), Bayer CropScience, 14/20 rue Pierre Baizet, BP 9163, F-69263 Lyon, Cedex 09, France; 2Institut National de la Recherche Agronomique, Unité Mixte de Recherche N° 6175, Service de Spectrométrie de Masse Pour la Protéomique, Nouzilly, France; 3Plant Research International, Wageningen University and Research Centre, PO Box 16, 6700 AA Wageningen, The Netherlands

Abstract Seed storage is often accompanied by a progressive loss of germination vigour and viability. In the present study, we have used Arabidopsis thaliana (L.) Heynh. seeds as a model, and carried out differential proteomics to investigate seed vigour. In our system, based on a controlled deterioration treatment (CDT), we compared seed lots treated for different time periods up to 7 days. Germination tests showed a progressive decrease of seed vigour depending on the duration of CDT. Proteomic analyses revealed that loss in seed vigour can be accounted for by protein changes in the dry seed and by an inability of the low vigour seeds to display a normal proteome during germination. Furthermore, the CDT strongly increased the extent of protein oxidation (i.e. carbonylation), which will in turn induce a loss of functional properties of proteins and enzymes and/or enhance their susceptibility towards proteolysis. These results highlighted essential mechanisms for germinative quality such as translational capacity and mobilization of seed storage reserves.

Introduction Before ageing irreparably leads to the death of the seed, the deterioration accumulated during storage is likely to affect its potential ability to germinate. This deterioration process occurs even under ideal storage conditions. The lifespan of seeds is determined by their genetic and physiological storage potential and by any deteriorating events that occur prior to or during storage, as well as by interaction with environmental factors. A recent investigation reported large differences in the response to storage of seeds descended from different plant species (Walters et al., 2005). We are interested in determining the molecular basis of seed storability in the dry state. The model plant Arabidopsis (Arabidopsis thaliana (L.) Heynh.) is a 324

Navie Ch34.indd 324

©CAB International 2006. Seeds: Biology, Development and Ecology (eds S. Navie, S. Adkins and S. Ashmore)

10/14/2006 11:40:43 AM

Seed Quality and Germination

325

very good reference species for this purpose, because it allows a molecular dissection of storage response. Indeed, disclosure of the Arabidopsis genome sequence (Arabidopsis Genome Initiative, 2000; Somerville and Koornneef, 2002) increased markedly our knowledge and understanding of the great complexity in regulation of plant growth and development. Genetic and global approaches such as transcriptomic profiling (Ogawa et al., 2003; Clerkx et al., 2004; Nakabayashi et al., 2005) have proved useful for the characterization of potential biomarkers of seed quality and germinative capacity. However, the functional components of a biological system are proteins and not genes or mRNAs. Thanks to the availability of genomic sequence information, the progress achieved in sensitive and rapid separation of proteins as well as their high-throughput identification by electrophoresis and mass spectrometry, proteomic approaches have opened up new perspectives to analyse the complex functions of model plants and crop species (Canovas et al., 2004). In this way, we have used proteomics to unravel the requirements in terms of RNA and protein synthesis for Arabidopsis seed germination (Gallardo et al., 2001, 2002a,b; Rajjou et al., 2004). In particular, these studies have revealed that proteins and mRNAs stored in dry mature seeds are sufficient for germination sensu stricto (Rajjou et al., 2004). In the present study we have used such proteomic tools and a seed deterioration treatment, known as CDT, which is presumed to mimic natural ageing (Clerkx et al., 2004). CDT is widely used as a vigour assay for numerous seed species and has been described for Arabidopsis seeds (Tesnier et al., 2002). We compared eight Arabidopsis seed lots treated for different time periods up to 7 days. A comparison of the dry seed proteome for each sample was carried out to reveal changes in the accumulation of specific proteins during the treatment. The proteome of 1-day-imbibed seeds was also characterized for all seed samples to analyse the behaviour of the treated seeds during the early steps of the germination process. Since the CDT is presumed to entail an oxidative stress, which can lead to the formation of oxidatively modified proteins, we also analysed the oxidized proteome in the treated seeds.

Materials and Methods Plant material and germination experiments Non-dormant seeds of Arabidopsis, accession Landsberg erecta (Ler), were used in all experiments. Germination assays were carried out at 25°C, with a 16 h light/8 h dark daily regime, as described in Rajjou et al. (2004).

Controlled deterioration test (CDT) The CDT was performed according to Tesnier et al. (2002). Seeds were briefly equilibrated at 85% relative humidity (RH) (20°C) and day 0 controls were immediately dried back at 32% RH. Treatment involved storing the seeds (at 85% RH) for various time periods (i.e. 0 h, 4 h, 16 h, 1 day, 2 days, 3 days, 5 days and 7 days) at 40°C. Seeds were then dried back at 32% RH (20°C) and stored at 4°C.

Navie Ch34.indd 325

10/14/2006 11:40:43 AM

326

L. Rajjou et al.

Preparation of total protein extracts and two-dimensional electrophoresis Total protein extracts were prepared from dry mature seeds and seeds at different stages of germination as described earlier (Rajjou et al., 2004; Job et al., 2005). Proteins, with an equivalent to an extract of 100 seeds, corresponding to about 200 µg protein for all samples, were analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as described earlier (Görg et al., 1987; Rajjou et al., 2004). Two-dimensional gels were stained with silver nitrate according to either Blum et al. (1987), for densitometric analyses, or Shevchenko et al. (1996), for the mass spectrometry analyses. Image analysis was carried out with the ImageMaster 2D Elite version 4.01 software (Amersham Biosciences).

Detection of oxidized proteins and western blotting Detection of oxidized proteins by carbonylation was performed by derivatization of protein extracts with 2–4 dinitrophenylhydrazine (DNPH) and immunological detection of the DNP adducts with monoclonal anti-DNP antibody (OxyBlot™ oxidized protein detection kit, Chemicon, France) as described earlier ( Job et al., 2005; Nyström, 2005).

De novo protein synthesis Labelled-proteins were synthesized in vivo by imbibing seeds in water for 1 day in the presence of [35S]methionine (1.85 MBq; ICN Biomedicals, S.A.R.L.). Protein synthesis was measured by trichloroacetic acid (TCA) precipitation of aliquots of reaction mixtures spotted on Whatmann GF/C filters; after ten washing steps in cold 5% TCA and 0.04 M sodium pyrophosphate, and two washing steps in absolute ethanol, filters were dried and counted for radioactivity in a liquid scintillation counter.

Protein identification by mass spectrometry Spots of interest were excised from 2D SDS-PAGE gels with sterile tips and put in 1.5 ml sterile tubes. Each spot was rinsed, reduced with 10 mM dithiothreitol (DTT), alkylated with 55 mM iodoacetamide and incubated overnight at 37°C with 12.5 ng/µl trypsin (sequencing grade; Roche Diagnostics) in 25 mM NH4HCO3. Analysis of tryptic peptides by tandem mass spectrometry (MS/MS) was performed on a nanoelectrospray ionization quadrupole time-of-flight hybrid mass spectrometer (Q-TOF Ultima Global; Waters Micromass, Manchester, UK) coupled with a nano-HPLC (Cap-LC; Waters), as described in Job et al. (2005). The peptide masses and sequences obtained were either matched automatically to proteins in a non-redundant database (NCBI) using the Mascot MS/MS Ions Search algorithm (http://www.matrixscience.com) or blasted manually against the current databases.

Navie Ch34.indd 326

10/14/2006 11:40:44 AM

Seed Quality and Germination

327

Results and Discussion Germination parameters of Arabidopsis seed samples The germination parameters of the presently studied seed samples are listed in Table 34.1. There was a dramatic decline in germinability of the seeds submitted to the CDT. Not only was the seed vigour affected, but there was also a strong reduction in seed viability as indicated by a marked decrease of the maximum germination percentage (Gmax). Proteome variation of dry mature seeds To explore the molecular mechanisms associated with the loss of seed vigour during accelerated ageing, a differential proteomic approach was used. To achieve this, total soluble proteins extracted from all seed lots were separated by 2D-PAGE. Following silver nitrate staining, protein patterns were analysed by image analysis. A typical gel is presented in Fig. 34.1. We first investigated the effect of the CDT on the dry seed proteome (i.e. from the proteome of seeds collected after conducting the treatment). Only a few protein spots showed reproducible variations in their accumulation level (Fig. 34.1). Our differential proteomic approach reveals that 12 protein spots were more abundant in the aged seeds and six protein spots were less abundant in the deteriorated seeds. These results highlight that protein modifications can occur during artificial seed ageing.

Characterization of oxidized proteins in the dry mature seeds Ageing in all organisms, notably in plants, is associated with oxidative stress (Bailly, 2004) that entails oxidation, by carbonylation, of specific proteins (Berlett and Stadtman, 1997; Toda, 2000). Since highly sensitive methods have been described Table 34.1. Germination parameters of Arabidopsis seed lots subjected to different periods of controlled deterioration treatment (CDT). Seed lot

t1% (h)

t10% (h)

t25% (h)

t50% (h)

t75% (h)

t90% (h)

Gmax (%)

0h 4h 16 h 1 day 2 days 3 days 5 days 7 days

39.6 41.1 41.0 43.0 45.2 47.9 59.8 102.7

40.9 42.5 42.5 44.5 46.8 49.8 62.5 –

41.9 43.6 43.5 45.7 48.0 51.1 64.9 –

43.2 45.1 45.0 47.4 49.7 53.2 – –

45.1 47.8 47.5 50.4 52.6 57.8 – –

49.0 63.1 57.3 64.0 – – – –

100 98 98 96 85 79 28 2

t1%, t10%, t25%, t50%, t75% and t90% are the times to reach 1%, 10%, 25%, 50%, 75% and 90% germination, respectively; Gmax = maximum germination after 1 week.

Navie Ch34.indd 327

10/14/2006 11:40:44 AM

328

L. Rajjou et al.

pI 3.0

5.0

5.5

MW (kDa)

5.9

6.6

8.7

293

82.3 304

64.1 4

305

320

376

50.0

321

212

302

41.0 253 146

7

322

34.7

26.4 308

254

18.4

311

255

312

13.3

Fig. 34.1. Detection of age-related protein alterations by 2D-PAGE. This figure shows a 2D gel of total soluble proteins from dry mature seeds after 7 days of CDT. Proteins were first separated by electrophoresis according to charge. Isoelectrofocusing (IEF) was carried out with protein samples with the equivalent to an extract of ~100 seeds, corresponding to ~200 µg protein for all samples. Proteins were then separated according to size by SDS-PAGE using 10% polyacrylamide gels. Proteins were visualized by silver nitrate staining. Black and white numbers indicate proteins with increased or decreased levels following the CDT, respectively.

for the detection of carbonylated proteins (Levine et al., 1990), we characterized the influence of the CDT on the oxidized proteome of Arabidopsis seeds (Fig. 34.2). Carbonylated proteins were identified by matching the 2,4-dinitrophenylhydrazone (DNP)-derivatized protein spots to master gel maps of Arabidopsis seed proteins (Gallardo et al., 2001, 2002a; Rajjou et al., 2004; http://seed.proteome.free.fr). The results revealed that protein carbonylation strongly increased in deteriorated seeds, indicative of the progressive accumulation of reactive oxygen species (ROS) during the CDT. The example presented in Fig. 34.2 shows that several polypeptides corresponding to the α- and β-subunits of the 12S cruciferins (legumin type seed storage proteins) are heavily carbonylated in the aged seeds. It must be stressed that in non-deteriorated seeds, carbonylation of

Navie Ch34.indd 328

10/14/2006 11:40:44 AM

Seed Quality and Germination

329

pI (a) MW 3.0 5.0 (kDa)

5.5

5.9

6.6

8.7

(b) Control seeds

MW (kDa)

(c) Deteriorated seeds

34.7

α Proteins

α

82.3 64.1

26.4

50.0 β

41.0

β 18.0

(d)

(e)

6.5

6.5

pI

34.7

26.4

α

α

18.4

(e) Carbonyls

34.7

26.4

β

β

13.3

18.0

Fig. 34.2. Increased protein carbonyl levels in β-subunits of 12S cruciferins after a CDT. Protein extracts were prepared from the dry mature seeds (control seeds) and seeds submitted to a CDT (deteriorated seeds) and analysed by 2D-PAGE. The portion of the 2D gels shown correspond to the window in panel (a). Protein silver stains (panels (b) and (c) ) and anti-DNP immunoassays (panels (d) and (e) ) are shown.

12S-cruciferin β-subunits was much lower than for the α-subunits (compare Figs. 34.2b and 34.2d). In marked contrast, the CDT entailed a strong increase in the extent of carbonylation of the 12S-cruciferin β-subunits, up to a level similar of that of the α-subunits (compare Figs. 34.2c and 34.2e). In addition to the 12S-cruciferin subunits, several other proteins ought to be oxidized in the aged Arabidopsis seeds (data not shown). In conclusion, the present data strongly support the finding that loss in seed vigour afforded by the CDT arises from overproduction of ROS associated with oxidative protein damage.

Proteome variation of germinating seeds The proteome of the artificially aged seed lots was also analysed after 1 day imbibition in water. This stage corresponds to the germination sensu stricto of Arabidopsis (Ler) non-deteriorated seeds (none of the seeds showed radicle protrusion at that time; see Table 34.1). This analysis revealed the proteome evolution of differentially aged seeds during imbibition. Among 54 protein spots presenting reproducible variations in their accumulation level, 34 were less abundant in germinating deteriorated seeds and 20 were more abundant. Functional categorization of these genes is presented in Fig. 34.3. It appears that several protein functions were affected by the CDT. One of the specific features observed from this clustering analysis is a larger abundance of storage proteins in germinating deteriorated seeds,

Navie Ch34.indd 329

10/14/2006 11:40:46 AM

330

L. Rajjou et al.

Transcription

(a)

(b)

Storage protein

Transport Storage protein

Cell division

Metabolism and energy Cell division

Translation and protein metabolism

Metabolism and energy

Translation and Protein metabolism Stress response and detoxification

Stress response and detoxification

Fig. 34.3. Distribution of functional categories of the seed proteins in germinating seeds whose accumulation level decreased (a) or increased (b) following the CDT using The Arabidopsis Information Resource’s (TAIR’s) Gene Ontology Resources.

a finding indicative of a correlation between the loss of seed vigour and storage mobilization ability. Another interesting feature concerns the apparent correlation between protein metabolism and translation, and the reduction of seed germinability induced by the CDT (Fig. 34.3). To get a direct insight into protein synthesis during germination, sensu stricto proteins that were newly synthesized in vivo following seed imbibition in water for 1 day were labelled in the presence of radioactive [35S]methionine. The control seed lot, which had a maximum germination of 100%, supported a very active [35S]methionine incorporation, testifying to a high translational activity during germination sensu stricto. As shown in Fig. 34.4, the extent of [35S]methionine incorporation declined dramatically in the deteriorated seed lots.

500

CPM/µg proteins

400

300

200

100

0 0 day

Navie Ch34.indd 330

3 days Seed samples

7 days

Fig. 34.4. Influence of the CDT on de novo protein synthesis. Seeds were incubated for 1 day in the presence of [35S]methionine. Protein synthesis was measured by trichloroacetic acid ( TCA) precipitation of aliquots of reaction mixtures spotted on Whatmann GF/C filters; after ten washing steps in cold 5% TCA and 0.04 M sodium pyrophosphate and two washing steps in absolute ethanol, filters were dried and counted for radioactivity (CPM) in a liquid scintillation counter.

10/14/2006 11:40:47 AM

Seed Quality and Germination

331

For example, seed lots that were deteriorated for 3 days presented an eightfold decrease in [35S]methionine incorporation compared with control seeds, although under these conditions the aged seeds still kept a high vigour with a maximum germination of about 80%. This result demonstrated that translation capacity can be an excellent criterion for the estimation of seed vigour, a finding that is in good agreement with the previous work on seed ageing in soybean (Glycine max (L.) Merr.) (Pillay, 1977). The seed lot that was deteriorated for 7 days had a maximum germination of ~2% and showed almost no translational activity. However, very low residual translation was detectable, suggesting that these seeds were not dead. The consequences of the observed reduction of protein synthesis can be diverse. This may affect the systems necessary for the maintenance, repair and normal resumption of metabolism, the efficiency of detoxification, the efficiency of the signalling pathways, and/or the production and secretion of several metabolites as well as plant hormones like gibberellins. Similar events occur during accelerated and natural ageing Uncertainty prevails as to why the CDT mimics natural ageing. This is a major concern of seed companies because, for practical reasons, they rely on the CDT and germination assays to predict seed storability (Delouche and Baskin, 1973). It is therefore of importance to compare the biochemical behaviour of seeds submitted to the CDT and of seeds that have been naturally aged. For that purpose, three naturally aged Arabidopsis seed lots were examined. Two of them, aged 7 and 8 years, presented a maximum germination of about 45% and 23%, respectively. A third one, aged 11 years, did not germinate at all after 14 days of imbibition. A proteome analysis revealed common features between the artificially and naturally aged seeds. Furthermore, the extent of protein carbonylation strongly increased during natural ageing, as also occurs in artificial ageing. Finally, protein translation capacity was strongly repressed in naturally aged seeds, a specific feature also observed with the CDT (Fig. 34.4). Our data thereby provides the first molecular indication supporting the usefulness of the CDT for prediction of seed storability.

Conclusions Proteomics provided an innovative and powerful tool for investigating the molecular mechanisms of seed vigour and seed viability during ageing. Changes in the regulation of protein synthesis, post-translational modifications and protein turnover are crucial determinants of age-related decline in the maintenance, repair and survival of the seed.

References Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408, 796–815. Bailly, C. (2004) Active oxygen species and antioxidants in seed biology. Seed Science Research 14, 93–107.

Navie Ch34.indd 331

10/14/2006 11:40:49 AM

332

L. Rajjou et al.

Berlett, B.S. and Stadtman, E.R. (1997) Protein oxidation in aging, disease and oxidative stress. The Journal of Biological Chemistry 272, 20313–20316. Blum, H., Beier, H. and Gross, H. J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99. Canovas, F.M., Dumas-Gaudot, E., Recorbet, G., Jorrin, J., Mock, H.P. and Rossignol, M. (2004) Plant proteome analysis. Proteomics 4, 285–298. Clerkx, E. J., El-Lithy, M.E., Vierling, E., Ruys, G. J., Blankestijn-De Vries, H., Groot, S.P., Vreugdenhil, D. and Koornneef, M. (2004) Analysis of natural allelic variation of Arabidopsis seed germination and seed longevity traits between the accessions Landsberg erecta and Shakdara, using a new recombinant inbred line population. Plant Physiology 135, 432–443. Delouche, J.C. and Baskin, C.C. (1973) Accelerated aging techniques for predicting the relative storability of seed lots. Seed Science and Technology 1, 427–452. Gallardo, K., Job, C., Groot, S.P.C., Puype, M., Demol, H., Vandekerckhove, J. and Job, D. (2001) Proteomic analysis of Arabidopsis seed germination and priming. Plant Physiology 126, 835–848. Gallardo, K., Job, C., Groot, S.P.C., Puype, M., Demol, H., Vandekerckhove, J. and Job, D. (2002a) Proteomics analysis of Arabidopsis seed germination: a comparative study of wild-type and GA-deficient seeds. Plant Physiology 129, 823–837. Gallardo, K., Job, C., Groot, S.P.C., Puype, M., Demol, H., Vandekerckhove, J. and Job, D. (2002b) Importance of methionine biosynthesis for Arabidopsis seed germination and seedling growth. Physiologia Plantarum 116, 238–247. Görg, A., Postel, W., Weser, J., Günther, S., Strahler, J.R., Hanash, S.M. and Somerlot, L. (1987) Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8, 122–124. Job, C., Rajjou, L., Lovigny, Y., Belghazi, M. and Job, D. (2005) Patterns of protein oxidation in Arabidopsis seeds and during germination. Plant Physiology 138, 790–802. Levine, L.R., Garland, D., Oliver, C.N., Amici, A., Climent, I., Lenz, A.-G., Ahn, B.-W., Shaltiel, S. and Stadtman, E.R. (1990) Determination of carbonyl content in oxidatively modified proteins. Methods in Enzymology 186, 464–478. Nakabayashi, K., Okamoto, M., Koshiba, T., Kamiya, Y. and Nambara, E. (2005) Genome-wide profiling of stored mRNA in Arabidopsis thaliana seed germination: epigenetic and genetic regulation of transcription in seed. The Plant Journal 41, 697–709. Nyström, T. (2005) Role of oxidative carbonylation in protein quality control and senescence. The EMBO Journal 24, 1311–1317. Ogawa, M., Hanada, A., Yamauchi, Y., Kuwahara, A., Kamiya, Y. and Yamaguchi, S. (2003) Gibberellin biosynthesis and response during Arabidopsis seed germination. The Plant Cell 15, 1591–1604. Pillay, D.T. (1977) Protein synthesis in aging soybean cotyledons: loss in translational capacity. Biochemical and Biophysical Research Communications 79, 796–804. Rajjou, L., Gallardo, K., Debeaujon, I., Vandekerckhove, J., Job, C. and Job, D. (2004) The effect of α-amanitin on the Arabidopsis seed proteome highlights the distinct roles of stored and neosynthesized mRNAs during germination. Plant Physiology 134, 1598–1613. Shevchenko, A., Wilm, M., Vorm, O. and Mann, M. (1996) Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Analytical Chemistry 68, 850–858. Somerville, C. and Koornneef, M. (2002) A fortunate choice: the history of Arabidopsis as a model plant. Nature Reviews Genetics 3, 883–889. Tesnier, K., Strookman-Donkers, H.M., van Pijlen, J.G., van der Geest, A.H.M., Bino, R. J. and Groot, S.P.C. (2002) A controlled deterioration test of Arabidopsis thaliana reveals genetic variation in seed quality. Seed Science and Technology 30, 149–165. Toda, T. (2000) Current status and perspectives of proteomics in aging research. Experimental Gerontology 35, 803–810. Walters, C., Wheeleer, L.M. and Grotenhuis, J.M. (2005) Longevity of seeds stored in a genebank: species characteristics. Seed Sciences Research 15, 1–20.

Navie Ch34.indd 332

10/14/2006 11:40:49 AM