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Development of the cotyledon cells during olive (Olea europaea L.) in vitro seed germination and seedling growth

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Abstract

The structural changes occurred in differentiating olive cotyledon cells into mesophyll cells are described. Using histological and immunocytological methods as well as microscopic observations, we showed that in the cells of mature embryo, large electron-dense proteins bodies (PBs) are surrounded by numerous oil bodies (OBs). After 3 days of in vitro germination, the presence of large PBs originated by fusion of smaller PBs was observed. It was also detected a close spatial proximity between PBs and OBs, likely as a reflection of interconnected metabolic pathways. Between the 3rd and the 12th day of germination, the formation of a large vacuolar compartment takes place accompanied by a decrease in the PBs and OBs number. This was coincident with a progressive decrease in the amount of the 11S-type seed storage proteins (SSPs), showed in situ and after Western blot analysis of crude protein extracts. After 26 days germination, the cellular organization became typical for a leaf mesophyll cell, with well-differentiated chloroplasts surrounding a large central vacuole. Our results suggest that the olive cotyledon storage reserves are mobilized gradually until the seedling becomes autotrophic. Moreover, the specific accumulation of storage proteins in the intravacuolar material suggests that these structures may operate as a shuttle for SSPs and/or products of their degradation into the cytoplasm, where finally they supply amino acids for the differentiating mesophyll cells.

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References

  • Alché JD, Jiménez-López JC, Wang W, Castro AJ, Rodríguez-García MI (2006) Biochemical characterization and cellular localization of 11S type storage proteins in olive (Olea europaea L.) seeds. J Agric Food Chem 54:5562–5570

    Article  Google Scholar 

  • Alvarez J, Guerra H (1985) Biochemical and morphological changes in protein bodies during germination of lentil seeds. J Exp Bot 36:1296–1303

    Article  CAS  Google Scholar 

  • Ashton FM (1979) Mobilization of storage proteins in seeds. Ann Rev Plant Physiol 27:95–117

    Article  Google Scholar 

  • Beevers H (1979) Microbodies in higher plants. Ann Rev Plant Physiol 30:159–193

    Article  CAS  Google Scholar 

  • Beisson F, Ferte N, Bruley S, Voultoury R, Verger R, Arondel V (2001) Oil bodies as substrates for lipolytic enzymes. Biochim Biophys Acta 1531:47–58

    PubMed  CAS  Google Scholar 

  • Bewley JD (1997) Seed germination and dormancy. Plant Cell 9:1055–1066

    Article  PubMed  CAS  Google Scholar 

  • Bewley JD, Black M (1994) Seeds: physiology of development and germination. Plenum, London

    Google Scholar 

  • Bhandari NN, Chitralekha P (1984) Degradation of protein bodies in germinating seeds of Brassica campestris L. var. sarson Prain. Ann Bot 53:793–801

    Google Scholar 

  • Cañas LA, Benbadis A (1988) In vitro plant regeneration from cotyledon fragment of the olive tree (Olea europaea L.). Plant Sci 54:65–74

    Article  Google Scholar 

  • Chen X, Schnell DJ (1999) Protein import into chloroplasts. Trends Cell Biol 9:222–227

    Article  PubMed  CAS  Google Scholar 

  • Eastmond PJ (2006) SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds. Plant Cell 18:665–675

    Article  PubMed  CAS  Google Scholar 

  • Fincher GB (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Ann Rev Plant Physiol Plant Molr Biol 49:305–346

    Article  Google Scholar 

  • Graham IA (2008) Seed storage oil mobilization. Ann Rev Plant Biol 59:115–142

    Article  CAS  Google Scholar 

  • Hara-Nishimura I, Hayashi M, Nishimura M, Akazawa T (1987) Biogenesis of protein bodies by budding from vacuoles in developing pumpkin cotyledons. Protoplasma 136:49–55

    Article  CAS  Google Scholar 

  • Harris N, Chrispeels MJ (1975) Histochemical and biochemical observations on storage protein metabolism and protein body autolysis in cotyledons of germinating mung beans. Plant Physiol 56:292–299

    Article  PubMed  CAS  Google Scholar 

  • Hayashi Y, Hayashi M, Hayashi H, Hara-Nishimura I, Nishimura M (2001) Direct interaction between glyoxysomes and lipid bodies in cotyledons of the Arabidopsis thaliana pedl mutant. Protoplasma 218:83–94

    Article  PubMed  CAS  Google Scholar 

  • Heneen WK, Karlsson G, Brismar K, Gummeson P-O, Marttila S, Leonova S, Carlsson AS, Bafor M, Banas A, Mattsson B, Debski H, Stymne S (2008) Fusion of oil bodies in endosperm of oat grains. Planta 228:589–599

    Article  PubMed  CAS  Google Scholar 

  • Herman EM (1987) Immunogold-localization and synthesis of an oil-body membrane protein in developing soybean seeds. Planta 172:336–345

    Article  CAS  Google Scholar 

  • Herman EM (1995) Cell and molecular biology of seed oil body proteins. In: Kigel J, Galili G (eds) Seed development and germination. Marcel Dekker, New York, pp 195–214

    Google Scholar 

  • Herman EM, Larkins BA (1999) Protein storage bodies and vacuoles. Plant Cell 11:601–613

    Article  PubMed  CAS  Google Scholar 

  • Huang AHC (1992) Oil bodies and oleosins in seeds. Ann Rev Plant Physiol Plant Mol Biol 43:177–200

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 277:680–685

    Article  Google Scholar 

  • Leprince O, Van Aelst AC, Pritchard HW, Murphy DJ (1998) Oleosins prevent oil-body coalescence during seed imbibition as suggested by a low-temperature scanning electron microscope study of desiccation-tolerant and -sensitive oilseeds. Planta 204:109–119

    Article  CAS  Google Scholar 

  • Lersten NR (2004) Flowering plant embryology. Blackwell, Oxford

    Book  Google Scholar 

  • Matile P (1982) Protein degradation. In: Boulter D, Parthier B (eds) Nucleic acids and proteins in plants. Encyclopedia of plant physiology, new series (Pirson A, Zimmermann MH, eds), vol 14A. Springer, Berlin, pp 169–188

    Google Scholar 

  • Müntz K (1998) Deposition of storage proteins. Plant Mol Biol 38:77–99

    Article  PubMed  Google Scholar 

  • Müntz K, Belozersky MA, Dunaevsky YE, Schlereth A, Tiedemann J (2001) Stored proteinases and the initiation of storage protein mobilization in seeds during germination and seedling growth. J Exp Bot 52:1741–1752

    Article  PubMed  Google Scholar 

  • Murphy DJ (2001) Biogenesis and functions of lipid bodies in animals, plants and microorganisms. Prog Lipid Res 40:325–438

    Article  PubMed  CAS  Google Scholar 

  • Murphy DJ, Cummins I (1989) Seed oil-bodies: isolation, composition and role of oil-body apolipoproteins. Phytochem 28:2063–2069

    Article  CAS  Google Scholar 

  • Murphy DJ, Vance J (1999) Mechanisms of lipid body formation. Trends Biochem Sci 24:109–115

    Article  PubMed  CAS  Google Scholar 

  • Ohto M, Stone SL, Harada JJ (2007) Genetic control of seed development and seed mass. In: Bradford KJ, Nonogaki H (eds) Seed development, dormancy and germination. Annu Plant Rev 27. Wiley-Blackwell, Oxford, pp 1–24

    Chapter  Google Scholar 

  • Otegui MS, Herder R, Schulze J, Jung R, Staehelin LA (2006) The proteolytic processing of seed storage proteins in Arabidopsis embryo cells starts in the multivesicular bodies. Plant Cell 18:2567–2581

    Article  PubMed  CAS  Google Scholar 

  • Parker J (1965) Stains for strands in sieve tubes. Stain Technol 40:223–225

    PubMed  CAS  Google Scholar 

  • Penfield S, Rylott EL, Gilday AD, Graham S, Larson TR, Graham IA (2004) Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in the dark, is independent of abscisic acid, and requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1. Plant Cell 16:2705–2718

    Article  PubMed  CAS  Google Scholar 

  • Poxleitner M, Rogers SW, Samuels AL, Browse J, Rogers J (2006) A role for caleosin in degradation of oil-body storage lipid during seed germination. Plant J 47:917–933

    Article  PubMed  CAS  Google Scholar 

  • Reibach PH, Benedict CR (1982) Biosynthesis of starch in proplastids of germinating Ricinus communis endosperm tissue. Plant Physiol 70:252–256

    Article  PubMed  CAS  Google Scholar 

  • Ross JHE, Sanchez J, Millan F, Murphy DJ (1993) Differential presence of oleosins in oleogenic seed and mesocarp tissues in olive (Olea europaea) and avocado (Persea americana). Plant Sci 93:203–210

    Article  CAS  Google Scholar 

  • Schlereth A, Becker C, Horstmann C, Tiedemann J, Müntz K (2000) Comparison of globulin mobilization and cysteine proteinases in embryonic axes and cotyledons during germination and seedling growth of vetch (Vicia sativa L.). J Exp Bot 349:1423–1433

    Article  Google Scholar 

  • Shewry PR, Napier JA, Tatham AS (1995) Seed storage proteins: structures and biosynthesis. Plant Cell 7:945–956

    Article  PubMed  CAS  Google Scholar 

  • Shutov AD, Vaintraub IA (1987) Degradation of storage proteins in germinating seeds. Phytochem 26:1557–1666

    Article  CAS  Google Scholar 

  • Tiedemann J, Neubohn B, Müntz K (2000) Different functions of vicilin and legumin are reflected in the histopattern of globulin mobilization during germination of vetch (Vicia sativa L.). Planta 211:1–12

    Article  PubMed  CAS  Google Scholar 

  • Torrent M, Geli IM, Ludevid MD (1989) Storage-protein hydrolysis and protein-body breakdown in germinated Zea mays L. seeds. Planta 180:90–95

    Article  CAS  Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Article  PubMed  CAS  Google Scholar 

  • Tzen JTC, Lie GC, Huang AH (1992) Characterization of the charged components and their topology on the surface of plant seed oil bodies. J Biol Chem 267:15626–15634

    PubMed  CAS  Google Scholar 

  • Tzen JTC, Cao YZ, Laurent P, Ratnayake C, Huang AHC (1993) Lipids, proteins, and structure of seed oil bodies from diverse species. Plant Physiol 101:267–276

    PubMed  CAS  Google Scholar 

  • Wang W, Alché JD, Castro AJ, Rodriguez-Garcia MI (2001) Characterization of seed storage proteins and their synthesis during seed development in Olea europea. Int J Dev Biol 45:S63–S64

    CAS  Google Scholar 

  • Wang J, Li Y, Lo SW, Hillmer S, Sun SSM, Robinson DG, Jiang L (2006) Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies. Plant Physiol 143:1628–1639

    Article  Google Scholar 

  • Wang W, Alche JD, Rodriguez-Garcia MI (2007) Characterization of olive storage proteins during seed development. Acta Physiol Plant 29:439–444

    Article  CAS  Google Scholar 

  • Wilson KA, Rightmire BR, Chen JC, Tan-Wilson AL (1986) Differential proteolysis of glycinin and β-conglycinin polypeptides during soybean germination and seedling growth. Plant Physiol 82:71–76

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by a grant of the “Consejería de Innovación, Ciencia y Empresa de la Junta de Andalucía” (project P06-AGR-01791). The Andalusian Regional Government also granted financial support to A.Z. and K.Z. The authors thank Ms. Conchita Martínez-Sierra for her excellent technical support.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Biochemistry, Cell and Molecular Biology of Plants, Estación Experimental del Zaidín, CSIC, Profesor Albareda 1, 18008, Granada, Spain

    Agnieszka Zienkiewicz, José Carlos Jiménez-López, Krzysztof Zienkiewicz, Juan de Dios Alché & María Isabel Rodríguez-García

  2. Department of Physiology and Molecular Biology of Plants, Nicolaus Copernicus University, 87-100, Toruń, Poland

    Agnieszka Zienkiewicz

  3. Department of Cell Biology, Nicolaus Copernicus University, 87-100, Toruń, Poland

    Krzysztof Zienkiewicz

Authors
  1. Agnieszka Zienkiewicz
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  2. José Carlos Jiménez-López
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  3. Krzysztof Zienkiewicz
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  4. Juan de Dios Alché
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  5. María Isabel Rodríguez-García
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Correspondence to María Isabel Rodríguez-García.

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Handling Editor: Hanns H. Kassemeyer

A. Zienkiewicz and J.C. Jiménez-López participated equally in this study and should both be considered as principal authors.

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Fig. S1

Histochemical study of the olive cotyledons during in vitro seed germination and seedling growth. Methylene blue stain (A – K) and Ponceau S stain (A’-K’) for LM. A and A’ – Mature dry seed: large and homogenous stained protein bodies (PBs), and no stained smaller oil bodies (OBs) filling-up the whole cytoplasm; strongly stained nucleus of irregular contour (N) is present in the middle of the cell. B and B’ - Imbibed seed: strongly and uniformly stained PB between numerous, non-stained OBs occupying the whole cell; the nucleus (N) shows similar features like in dry, mature seed. C and C’ – Seed after 6 hours of germination: larger, PB showing different grade of staining; apparently decrease in the number of OBs is visible; the nucleus (N) volume increase and exhibits the features of an active interphase nucleus with a nucleolus (No). D and D’ - Seed after 2 days of germination: the cell organization not changes in relation to previous stage. Enlarged, stained (PBs) with small rounded non-stained areas (circles) occupying the most volume of the cell; (OBs) surrounding the protein bodies are not stained; the nucleus (N) with nucleolus is localized in the middle of the cell. E and E’ - Seed after 3 days (70 h) of in vitro germination: a few of large, stained PBs occupy most of the cell volume. Strongly stained, rounded small areas are observed inside of PBs; the nucleus (N) appears sideways displaced. F and F’ – Seed after 3 days and half (80 h) of germination: two, larger slightly stained PBs are filling up the cell. The rest of the cytoplasm, containing well-organized sideways nucleus (N) and OBs, localized mainly on the cell periphery. G and G’ – 4 days of in vitro germination: one large central protein body, slightly stained occupying the most cell volume is observed in; it is surrounded by a thin sheet of cytoplasm with an strongly stained nucleus (N). H and H’ - 8 days of seedling growth: the most significative change respect to earlier stage is the new heterogenous structure of the matrix of the central compartment. I to I’ –12 days of seedling growth: the cell present a large central vacuole (V) containing numerous, strongly stained inclusions (arrows) after Ponceau S staining; plastids (P) containing starch grains are also observed on the cell periphery. J and J’ - 18 days of germination: developing chloroplasts (P), containing starch grains are localized around the central vacuole (V); only a few dark inclusions (arrows) are yet founded inside the vacuole. K and K’ - 26 days of seedling growth: large, well developed chloroplasts (Chl) are localized around the large, central vacuole (V); the sideway nucleus (N) shows numerous dark inclusions, positive to protein staining. Scale bars = 10 μm (PDF 751 kb)

Fig. S2

Quantitative analysis of the cell size, PB and OB area and number during cotyledon differentiation. Average area of the cotyledon cell (A), PBs (B) and OBs (C) during the different germination stages. Values represent the average (μm2) of thirty measurements of the surface from three replicates; the bars indicate the standard error of the mean. Average number of PBs (B’) and OBs (C’) per cell obtained from thirty measurements in three replicates; the bars indicate the standard error of the mean. 1- Imbibed seeds, 2–6 h of germination, 3–3 days of germination, 4–4 days of germination, 5–18 days of plant growth, 6–26 days of plant growth. A - The section dimension of cotyledon cells slightly increased (between 1800 μm2 and 3000 μm2) during seed germination. The cells reached the larger size at the later steps of seed germination, just before the end of differentiation. B - The major changes in the single PBs area per cell, from 100 μm2 to 1700 μm2, are observed during first 4 days of germination. This increase started at the embryo imbibition and progressed until the 4th day of in vitro germination, with the augment of PBs taking place between the 3th and 4th day of germination when a single central compartment was formed. After this stage no more PBs were recognizable as such structures. B’ - The highest number of PBs was observed in the mature and imbibed seed. The enlargement of PBs during the differentiation of the cotyledon cells was accompanied by an apparent reduction of their number per cell. During early steps of seed germination a gradual and continuous decrease in the number of PBs occurred. Such tendency was observed until the 4th day of germination, when only one central compartment is formed in the cell. C - The average size of a single OB per cell was the largest in mature and imbibed seed. As germination proceeded a gradual decrease in OBs size was observed, from 8 μm2 to 1,5 μm2 at the 4th day of culture. After this day the presence of OBs is very scant. C’ - The analysis of OBs number per cell, during in vitro seed germination, showed a gradual decrease of their number. They were shown to be the most numerous in the mature (not shown) and imbibed seed. As germination started, a continuous reduction of OBs pool occurred. This decrease was observed during all further steps of seed germination, until the complete lack of OBs after 9 days of culture (PDF 168 kb)

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Zienkiewicz, A., Jiménez-López, J.C., Zienkiewicz, K. et al. Development of the cotyledon cells during olive (Olea europaea L.) in vitro seed germination and seedling growth. Protoplasma 248, 751–765 (2011). https://doi.org/10.1007/s00709-010-0242-5

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