16637409

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Original article

Variations in storage protein and carbohydrate levels during

development of avocado zygotic embryos

Carolina Sánchez-Romero

a

_{, Rosa Perán-Quesada}

a

_{, Araceli Barceló-Muñoz}

a

_,

Fernando Pliego-Alfaro

b,

_*

a_{C.I.F.A., 29140 Churriana, Málaga, Spain}

b_{Departamento de Biología Vegetal, Universidad de Málaga, Campus de Teatinos s/n, 29071 Málaga, Spain}

Received 27 March 2002; accepted 24 June 2002

Abstract

Variations in carbohydrates and proteins were monitored during avocado (Persea americana Mill.) zygotic embryo development and correlated with growth parameters in order to define specific markers characterizing distinct embryogenic phases. Hexose (glucose and fructose) levels were initially high and declined as embryo development advanced reaching the lowest levels in completely mature embryos. Sucrose and starch evolution showed an opposite trend with a progressive increase during embryo growth. The beginning of the maturation phase could be identified by a switch in the carbohydrate status from high hexose/sucrose ratio to low hexose/sucrose ratio. Storage protein accumulation began at early cotyledonary stages (7–8 mm), increasing significantly in the maturation phase where they represented 83% of total proteins. Mature embryos (38–40 mm) contained albumins, globulins and glutelins, albumins being the predominant and most heterogeneous fraction. Storage protein accumulation occurred in a sequential and specific way suggesting a possible role as indicators of embryo development. The complete maturation stage could be characterized by the synthesis and accumulation of a 49 kDa albumin. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Avocado; Hexose/sucrose ratio; Maturation; Soluble sugars; Starch; Storage proteins; Zygotic embryo

1. Introduction

Embryogenesis is a complex process that begins with a single cell and results in the formation of a mature embryo which will give rise to a plant in full multicellularity, sexuality and structure[24]. Embryo development involves repeated mitosis, establishment of polarity, histodifferentia-tion and accumulahistodifferentia-tion of complex metabolites and storage reserves [24]. These processes occur in a successive way suggesting an underlying developmental program, whose components come into play in a sequential order [26]. In

this ordered sequence, a number of stages can be differen-tiated: cell division, differentiation and maturation[31].

Critical to embryogenic development is the maturation phase since a major change occurs during this period: a switch from a regional and cell-specification program to a storage accumulation program in order to prepare the embryo for post-embryonic development [33]. Moreover, during maturation, many of the unique processes required for embryo formation occur: the synthesis and accumulation of nutrient reserves, the suppression of precocious germi-nation, the acquisition of desiccation tolerance and, in some species, the induction of dormancy[10]. Reserve products accumulation plays an essential role in seedling survival providing compounds that are utilized by the germinating embryo until the development of autotrophy[14]. The main storage products are normally carbohydrates, lipids and proteins. While carbohydrates and lipids are used as a source of energy and carbon skeletons[16], proteins provide carbon, nitrogen and sulfur[18]. However, in maturation, not only are the deposited products important, but soluble

Abbreviations: Bes, N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic

acid; BSA, bovine serum albumin; EDTA, ethylendiaminetetraacetic acid; FW, fresh weight; G6P-DH, glucose-6-phosphate dehydrogenase; HK, hexokinase; Mes, 2-(N-Morpholino)ethanesulfonic acid; MM, molecular mass; PGI, phosphoglucose isomerase; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel electrophoresis; Tris, Tris-(hydroxymethyl) aminomethane

* Corresponding author.

E-mail address: [email protected] (F. Pliego-Alfaro).

www.elsevier.com/locate/plaphy

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sugars, considered as minor storage products and a source for starch synthesis, could also act in regulating metabolism and gene expression[8].

Most physiological studies related to embryogenesis have been carried out in orthodox embryos. However, avocado (Persea americana Mill.) and a number of inter-esting tropical crops are recalcitrant embryos and do not fit the developmental pattern of orthodox seeds. Major differ-ences between both types of embryos are related to matu-ration phase e.g., recalcitrant embryos are generally much larger and fleshier than orthodox embryos, do not tolerate maturation drying and lack a period of developmental arrest [22].

The aim of this study was the establishment of an embryogenic developmental pattern for avocado, with spe-cial attention to the maturation phase. Variations in carbo-hydrates and proteins along the growth of zygotic embryo were correlated with growth parameters. Defining specific markers characterizing embryogenic phases, and what is more important, identifying changes associated to func-tional differentiation, would be an interesting instrument for physiological and genetic studies and could be used for evaluating zygotic and somatic embryo development in vitro.

2. Results

2.1. Growth and water content during zygotic embryo development

Embryo fresh weight (FW) increased progressively until the embryo reached 25 mm in length (Fig. 1); from 25 mm to the end of development, fresh weight increase was more pronounced. Zygotic embryo reached its final size approxi-mately 40 weeks after anthesis (25.47 g fresh weight and 40 mm in length). Dry weight evolution was similar to fresh weight trend.

Water content did not vary for embryo sizes in the range 10–19 mm. From this length onwards, it declined slightly within the range 19–25 mm and drastically in embryos of 25–34 mm in length. Afterwards, no significant variations were found.

2.2. Soluble carbohydrate content

Glucose and fructose contents showed similar trends, although glucose levels were always lower than fructose levels (Fig. 2). Glucose and fructose contents remained relatively constant during the early cotyledonary stages (7–8 mm), reaching the highest levels in embryos 15 mm long. As embryo development progressed, a gradual de-crease was observed, reaching the lowest values (0.3 mg g–1_{fw) in mature embryos. An opposite trend was}

observed in sucrose (Fig. 2). Low levels of sucrose were detected initially with the minimum value (1.25 mg g–1_fw)

in embryos 15 mm long. Further development was charac-terized by an increase in sucrose content, reaching the highest value in completely mature embryos.

Hexose/sucrose ratio was initially high due to the high hexose levels and low sucrose level observed in embryos 7–8 mm long (Fig. 2). In embryos 25 mm long, a switch in the hexose/sucrose ratio took place due to a decrease in hexose levels and an increase in sucrose level. The trend continued in the following developmental stages reaching the lowest hexose/sucrose ratio in embryos 38–40 mm long.

2.3. Starch content

Starch content increased during embryo development towards maturity. In the early cotyledonary stage (7–8 mm), starch content was moderate and from 15 mm size onwards, it increased significantly (Fig. 2). The highest level (10.24 mg g–1_{fw) was observed in fully developed}

em-bryos.

Fig. 1. Fresh weight, dry weight and water content in avocado (cv. Hass) zygotic embryos at different developmental stages. Data represent means±SE for 6–9 embryos for each size.

Fig. 2. Soluble sugars (glucose, fructose and sucrose) and starch contents during avocado (cv. Hass) zygotic embryo development. Data represent means±SE for three determinations from three different extracts (nine measurements).

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2.4. Storage proteins: identification and classification

Identification of storage proteins was inferred from their presence in protein bodies isolated from cotyledons of completely mature zygotic embryos (38–40 mm).

SDS-PAGE analysis of the protein body fraction (Fig. 3) showed that zygotic embryos contained storage proteins with molecular masses between 26 and 51 kDa. In contrast to the pattern shown by the complete zygotic embryo, no protein larger than 51 kDa could be observed. The electro-phoretic profile of embryo storage proteins showed two major components, e.g. 49 and 51 kDa, as well as two minor groups, 26–35 kDa and 40–48 kDa.

Storage proteins have been classified into four groups according to their solubility properties: albumins, globulins, prolamins and glutelins [34]. SDS-PAGE of each fraction (Fig. 3) showed that the mature embryo presented albumins, globulins and glutelins. No band appeared in the prolamin fraction.

Albumins displayed a heterogeneous pattern with two major bands of 49 and 51 kDa. Globulins only showed a band of 34 kDa whereas in the glutelin fraction, a low number of proteins with a main component of 51 kDa were detected.

Densitometric analysis showed that storage proteins represented 83% of total proteins in the mature embryo (Table 1). Albumins were the largest fraction accounting for 54% of storage proteins while glutelins and globulins represented 37.6% and 6.1%, respectively. The 51 and 49 kDa proteins accounted for 18.3% and 16.7%, respec-tively, of total storage proteins.

2.5. Protein accumulation pattern during zygotic embryo development

SDS-PAGE analysis of embryo total proteins (Fig. 4) showed a similar proteic composition in embryos at the earlier developmental stages (7–8, 14–16 and 24–26 mm long) where a series of proteins with molecular masses between 26 and 79 kDa could be observed. According to the results previously shown, the 40–48 kDa group could be identified as storage proteins and classified as albumins. This group of proteins appeared in embryos at the earliest developmental stage studied (7–8 mm) and was kept as the only storage proteins until the embryo reached 34–36 mm in length. From this stage onwards, the protein pattern signifi-cantly changed, appearing a protein of 51 kDa and a new protein group, within the range 26–35 kDa. The 51 kDa protein and part of the 26–35 kDa protein group, were also present in the isolated protein bodies and, therefore, could be considered as storage proteins. In the most advanced developmental stage, corresponding to embryos of 38–40 mm and physiologically mature, the intensity of the 51 kDa protein increased significantly and a new band appeared corresponding to an albumin of 49 kDa.

In conclusion, a series of storage proteins initiated their accumulation at the earlier developmental stages and

main-Fig. 3. SDS-PAGE analysis of storage proteins from avocado (cv. Hass) zygotic embryos. Lanes 38–40, total proteins of mature (38–40 mm) embryos; A, albumin fraction; G, globulin fraction; Pr, prolamin fraction; Gl, glutelin fraction; PB, total storage proteins; MM molecular mass standards. Numbers indicate molecular masses of protein markers in kDa. Proteins were resolved on a 10% SDS polyacrylamide gel and stained with Coomassie Brilliant Blue. Equal amounts of proteins were loaded in fraction lanes (A, G, Pr, Gl) while in samples from mature embryos and protein bodies, the amount of protein loaded was fourfold. Gel is representative of three independent gels carried out with samples from different embryos.

Table 1

Storage protein distribution in mature zygotic embryos. Values are means±SE of six densitometric scans from three independent gels carried out with samples from different embryos

% of total proteins % of storage proteins

Storage proteins 83.0±2.2 –

Albumins – 54.0±2.2

Globulins – 6.1±0.6

Prolamins – 0

Glutelins – 37.6±5.0

51 kDa – 18.3±4.5

49 kDa – 16.7±3.9

Fig. 4. SDS-PAGE analysis of total proteins of avocado (cv. Hass) zygotic embryos at different developmental stages. Numbers indicate embryo length in mm, MM are molecular mass standards and arrowheads show storage proteins. Numbers indicate molecular masses of protein markers in kDa. Proteins were resolved on a 10% SDS polyacrylamide gel and stained with Coomassie Brilliant Blue. Equal amounts of proteins were loaded in each lane. Gel is representative of three independent gels carried out with samples from different embryos.

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tained their presence throughout embryo development. Other proteins (51 kDa) accumulated coinciding with a drastic decline in the water content while there was another protein (49 kDa) whose expression was associated with the latest maturation stage.

3. Discussion

Avocado embryo development has been divided into different phases taking as basis histochemical observations [20]. According to this study, active cell division occurs from fruit set until the embryo reaches 3–5 mm in length, the histodifferentiation phase is completed in embryos 16–18 mm long and, after a transition phase without notice-able morphological changes, the beginning of the matura-tion phase takes place in embryos 23–25 mm long being identified by a significant increase in the number and size of starch grains as well as the appearance of protein bodies.

In the present study, it is shown that total soluble carbohydrate content and partitioning between particular sugars are remarkably influenced by embryo developmental stage [12]. According to its role as source of carbon and energy, high glucose levels quantified at early developmen-tal stages of avocado zygotic embryo could be meeting the high metabolic demand typical of this period where active cell division and differentiation take place[29]as stated by Perán-Quesada[20] using histochemical techniques. Later on, on embryos larger than 15 mm, the decrease in glucose content observed could be attributed to its utilization as a source for the synthesis of sucrose and starch that begin their accumulation at this stage showing a similar trend to that observed by Lipavská et al. [12] in Norway spruce somatic embryos.

Sucrose importance in embryo development is beyond any direct role as the main substrate for sink metabolism or the main form of translocated carbon. A significant regula-tory and integrative function has been suggested for sucrose via modulation of gene expression, protein turnover or its potential to modulate the activities of enzymes involved in its own synthesis, cleavage or metabolism[4]. In avocado embryo development, evolution of sucrose levels agrees with a possible role inducing the storage pathway as suggested by Weber et al.[31]. The significant increase in sucrose content observed in 25 mm long embryos coincides with the beginning of the maturation phase characterized by drastic fresh and dry weight increases, a decline in water content as well as the massive accumulation of starch grains and the appearance of protein bodies[20]. However, it has been postulated that more than the sucrose content itself, it is the sugar status that provides signals regulating metabo-lism [32]. During early developmental stages, until the embryo reaches 15 mm in length, avocado zygotic embryos show the high hexose/sucrose ratio characterizing the prestorage phase[32]. This special sugar status, with a high level of hexoses, seems to be necessary for promoting

growth[31]. The starting of the maturation phase is marked by a switch from a high hexose status to a sucrose based carbohydrate status that initiates storage-associated differ-entiation [30]. This change takes place in 25 mm long avocado embryos and coincides with other changes previ-ously indicated, e.g. starch grain accumulation and appear-ance of protein bodies[20]confirming the beginning of the maturation phase. During maturation of Norway spruce embryos, sugar partitioning between sucrose and hexoses changes in favor of sucrose [28]. In avocado, this trend is followed and the completed maturation stage shows the lowest value of the hexose/sucrose ratio.

In relation to starch content, the observed correlation between sucrose and starch levels would agree with the hypothesis that starch accumulation seems to be dependent on a high sucrose concentration in the cotyledon[32]. This dependence can be attributed to two possible reasons e.g., sucrose is the primary source for starch synthesis[16]and sucrose might have a stimulating effect on starch accumu-lation[32].

Total protein variations throughout avocado embryo development reveal an important role of storage proteins. This result agrees with previous reports signaling storage proteins as indicators of embryo developmental phases[27]. Storage protein analysis shows that albumins are the pre-dominant fraction in the avocado zygotic embryo. Although albumins have been considered for a long time as metabolic proteins, lately they are being considered as storage proteins due to their wide distribution and their amino acidic composition, rich in amide content [23]. Globulins have been generally considered as the main storage proteins in dicotyledonous[17]. However, in avocado, they constitute a fraction quantitatively not important and the only protein present does not have a differential behavior during embryo development. The role of glutelins is not clear since molecular mass of the predominant band coincides with that of an albumin. However, it is interesting to notice that other recalcitrant embryos, such as Quercus ilex and Quercus

robur, also deposit glutelins as storage proteins[1]. In relation to the temporal accumulation pattern of storage proteins, they begin to accumulate in embryos of small size (7–8 mm), although as found in oil palm[15]and interior spruce[5], the larger part is deposited at the end of the embryo growth period. The presence of storage proteins in embryos at earlier developmental stages has been de-scribed in some angiosperms [3,25], and particularly in

Citrus, they can be found in embryos at the globular stage

[9].

The presence of specific storage proteins as well as their relative importance shows noticeable changes during avo-cado embryo development. The presence of specific storage proteins in determined embryogenic stages has been previ-ously reported by distinct authors [5,23]. Raghavan [23] differentiates storage proteins appearing at early develop-mental stages (in avocado embryos it would correspond to 40, 45 and 48 kDa albumins), proteins accumulating when

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cell division in developing cotyledons has finished (in avocado it would correspond, although in a more advanced stage, to the 51 kDa protein) and finally, specific proteins deposited at the late maturation phase (in avocado it would be the 49 kDa albumin appearing only when the embryo has totally completed its development). The sequential biosyn-thesis and accumulation of specific storage proteins make them very useful markers of developmental processes and show that embryo storage proteins are under a strict developmental control[6].

As carbohydrates provide carbon skeletons needed for protein biosynthesis[31], it is clear that a connection must exist between carbohydrates and protein metabolism. It has been suggested the existence of key genes involved in carbon partitioning between starch and proteins[16].

In avocado zygotic embryos, variations in carbohydrate status seem to indicate the beginning of the reserve depo-sition phase whereas accumulation of specific proteins marks the termination of this process and, consequently, the achievement of a physiological maturity which capacitates the embryo to face the germination process. Therefore, two steps seem to have a key importance in the final attainment of the embryogenic process, e.g. the switch in the carbohy-drate status and the deflection of carbon skeletons from starch to protein synthesis. Finding mechanisms and factors controlling these critical steps are key points to understand embryogenic development and are being revealed as ex-tremely useful tools to optimize zygotic and somatic em-bryos culture conditions.

4. Methods

4.1. Plant material

Avocado (Persea americana Mill.) zygotic embryos were used as plant material. Trees of cv. Hass were growing in a monovarietal orchard at CIFA Churriana (Málaga) under an open pollination regime. Cultivation practices were those considered standard for commercial avocado orchards. Fruits of uniform size were selected at random from different trees. Embryos, excised from seeds, were classified according to their developmental stage by using growth parameters: length and weight. The classified mate-rial was immediately frozen in liquid nitrogen and stored at –80 °C. For protein bodies’ isolation, fresh fully devel-oped embryos (38–40 mm) were used. Dry weight was determined after heating zygotic embryos at 70 °C for 48 h.

4.2. Soluble sugar quantification

Soluble sugars were extracted according to Coombs et al. [2]. After pulverizing in liquid nitrogen, the frozen sample was ground into ice-cold 1 M HClO₄ and centrifuged at

35,000×g for 10 min. The supernatant obtained was

imme-diately neutralized to pH 7 with 3 M K₂CO₃. KClO₄ precipitated was removed by filtration through Whatman GF/C paper. All operations were carried out at 4 °C. Glucose, fructose and sucrose were quantified following the method of Outlaw and Tarczynski [19] modified by Mercado-Carmona[13], e.g. 1 ml of assay cocktail solution (0.1 M Bes buffer (pH 6.9), 5 mM MgCl₂, 1 mM ATP, 0.4 mM NADP, 0.5 mM EDTA and 0.02% (w/v) BSA) and 30µl of the sample were mixed and absorbance at 340 nm was obtained. Ten microliters of a hexoquinase (HK)/glucose-6-phosphate dehydrogenase (G6P-DH) solu-tion (5010 nkat HK and 2505 nkat G6P-DH) and the same amount of a phosphoglucose isomerase (PGI) solution (5511 nkat PGI) were added sequentially after 10 min of incubation at room temperature in each case. Finally, 10µl of an invertase solution (133,600 nkat invertase) were added and kept incubated for 30 min. Absorbance increases due to each addition were recorded at 340 nm.

4.3. Starch quantification

Starch digestion to glucose was carried out following, with slight modifications, the method of Coombs et al.[2]. The pellet obtained in the percloric extraction and stored at –80 °C was washed three times with 0.2 M Mes buffer (pH 4.5) and autoclaved for 60 min at 121 °C and 0.1 MPa to gelatinize starch. After cooling, the sample was incubated at 55 °C for 2 h with 66,800 nkat amyloglucosidase and 33,400 nkat amylase and centrifuged for 10 min at 35,000×g and 4 °C. Glucose in the supernatant was

enzy-matically quantified as above. Starch content was deter-mined by the amount of glucose generated using a set of insoluble starch standards treated as above.

4.4. Protein determination

Protein bodies were isolated from cotyledons of com-pletely mature embryos by differential centrifugation in glycerol according to Huang [7]. Total proteins were ex-tracted by homogenizing zygotic embryos with 62.5 mM Tris–HCl (pH 6.8), 2% (w/v) SDS, 4 M urea and 5 mM 2-mercaptoethanol. Storage protein extractions were carried out using protein body pellets. Fractionation of storage proteins was carried out following, with some modifica-tions, the method of Yamagata et al.[34]. Storage proteins were extracted sequentially by homogenizing the protein bodies’ pellet with 10 mM Tris–HCl (pH 7.5) containing 1 mM EDTA (solvent A) to extract albumins; 0.5 M NaCl as supplement to solvent A for globulins; 60% (v/v) n-propanol as supplement to solvent A for prolamins, and 62.5 mM Tris–HCl (pH 6.8), 2% (w/v) SDS and 4 M urea for glutelins and residual proteins. Protein content was deter-mined according to Peterson[21]. Previous to the assay, the samples were precipitated with RNA-TCA to remove inter-fering substances, e.g. 25µl of 5 mg ml–1 _{soluble yeast}

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RNA and 100µl of 72% (w/v) TCA were added to 1 ml of protein sample. After mixing and leaving on ice for 45 min, the samples were centrifuged at 27,000×g and 4 °C for

10 min. BSA was used as a standard.

4.5. SDS-PAGE

Proteins were separated by SDS-PAGE according to Laemmli [11] on a 10% acrylamide (resolving) and 5% acrylamide (stacking) gel. Protein samples were prepared for electrophoresis by incubation with sample buffer (12 mM Tris–HCl (pH 6.8), 5% (v/v) glycerol, 0.4% (w/v) SDS, 2.88 mM 2-mercaptoethanol and 0.02% (w/v) bro-mophenol blue) at 100 °C for 5 min. Proteins were stained with Coomassie Brilliant Blue-R 250. Molecular masses of proteins were determined relative to marker proteins (SDS-7 Sigma, Missouri). The relative band intensities were analyzed densitometrically by a computer-assisted program (area scan method using the Scion Image Program).

Acknowledgements

This work was supported by the Comision Interministe-rial de Ciencia y Tecnología, Spain (grant no. AGL 2000-2003, C03-03).

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