Proteomic analysis of stipe explants reveals differentially expressed proteins involved in early direct somatic embryogenesis of the tree fern Cyathea delgadii Sternb
Introduction
Somatic embryogenesis (SE) is the developmental reprogramming of plant somatic cells toward the embryonic pathway, enabling non-zygotic cells to form embryos. On the molecular level, the embryonic developmental pathways leading to reprogramming have been divided into three phases: dedifferentiation, expression of totipotency, and commitment (induction stage) [1]. The expression of SE is the second developmental stage of this process, in which the somatic embryos begin to appear and develop. During the last decade, there has been significant progress in understanding the interactions between key factors initiating SE [1], [2]. However, whereas intensive research has been conducted in SE in spermatophytes for many years, in plant species from Monilophyta clad this has not been studied yet. Cyathea delgadii Sternb. is the first fern for which SE has been described [3]. In parallel with the elaboration of a quick and effective protocol for the micropropagation of the tree fern, we propose a unique model for SE study. In this model system, both the induction and development of somatic embryos occur on hormone-free medium [4]. A long-term dark treatment of donor plantlets is the key factor for SE induction in C. delgadii [3]. Moreover, we showed that the epidermal cells of stipe explants acquire an embryogenic competence during 10 days of initial culture. The explants are able to produce somatic embryos originating from single cells and to achieve a multicellular linear stage in the next few days [4]. The described model system could be very helpful in the discovery/explanation of the mechanisms involved in early SE.
Microscopic analysis has been used to explore morphogenetic events occurring during the initiation of direct or indirect SE [5], [6], [7], [8], [9], and somatic embryo formation by unicellular or multicellular pathways [7], [8]. Such events as the accumulation of storage products (starch, proteins and lipids), and the increase in cell wall thickness and in cytoplasm density are hallmarks of the induction phase of SE and subsequent somatic embryo differentiation [6], [9], [10], [11], [12], [13]. In ferns, no structural changes have been described during SE, except for some cytological features of the zygotic embryogenesis of the fern Ceratopteris richardii and C. thalictroides [14], [15].
To observe a wide range of changes in protein profiles accompanying the direct physiological process, the proteomic approach seems to be the most suitable [1]. Proteomic analyzes are a powerful method to study biochemical changes during plant developmental processes and its responses to various biotic and abiotic stresses. A comprehensive protein expression profiling can be performed using a two-dimensional gel electrophoresis (2-DE) technique enabling high resolution protein separation. 2DE is combined with the mass spectrometry method to identify differentially expressed proteins. The 2DE technique is the most suitable method to study plant species where the genomes are still unsequenced [16]. Moreover, quantitative and qualitative changes in the level of specific proteins can be detected by creating a map of biomarkers [17]. Some studies have been conducted using this tool to discover the mechanisms that control and regulate the induction and expression of SE [18]. However, most 2DE reports have been applied to investigate proteomic differences in indirect SE by comparison of embryogenic and non-embryogenic calli of Cupressus sempervirens L. [19], Vitis vinifera L. [20], Medicago truncatula Gaertn. [21], [22], Cyclamen persicum Mill. [23], Crocus sativus L. [24], Eruca sativa Mill. [25], Cyphomandra betacea (Cav.) Sendtner [26], Zea mays L. [27], [28], Elaeis guineensis Jacq. [29] and Liriodendron hybrid [30]. Certain proteomic studies have also focused on differentially expressed proteins during the development of somatic embryos [18]. Although many plant species have been regenerated via SE, little information is available on the proteomic events associated with an early developmental stage of direct SE [11], [31], and this is completely unexplored in cryptogamic plants. Therefore, the aim of the present study was to identify structural changes and differentially expressed proteins in early SE induced on stipe explants of the tree fern C. delgadii.
Section snippets
Plant material
Somatic embryo-derived sporophyte culture was obtained according to a previously published protocol [4]. The stock plantlets were maintained on a medium containing half-strength Murashige and Skoog’s [32] macro- and micro-nutrients (1/2MS) supplemented with 2% (w/v) sucrose, solidified with 0.7% plant agar (Duchefa Biochemie); pH = 5.8. The sporophyte culture was kept in darkness in a growth chamber at 24 ± 1 °C.
For SE induction, a 4-month-old culture of etiolated sporophytes that had developed 2-3
Cyto-morphological description of stipe explants used for proteomic analysis
Microscopic analysis of control C. delgadii stipe explants (Fig. 1a) which were freshly excised from juvenile sporophytes cultured in darkness for 4 months showed that their epidermal and cortical cells contained strongly vacuolated protoplasts. They were surrounded by uniformly thin cell walls (Fig. 1b-c). Their well-developed vacuoles contained a small number of grains of a strongly osmiophilic substance of unknown character. The layer of the cytoplasm was relatively thin and its most
Cyto-morphological changes involved in direct somatic embryogenesis
Direct SE has been described for many plant species, however, for some of these a limited proliferation of the proembryogenic zone before somatic embryo differentiation has been reported [5], [6], [7], [8], [9]. These meristematic cells have been proved to be proembryogenic cells which have the capacity to form somatic embryo via multicellular or unicellular pathways [7], [8]. Sometimes this process of regeneration is considered an intermediate between direct and indirect embryogenesis [6]. In
Conclusions
Proteomic analysis revealed that several proteins involved in the carbohydrate metabolism, protein and amino acid metabolism, anti-oxidative stress response and cell organization as well as transport, fatty acid and purine metabolisms were associated with direct SE in C. delgadii. The differentially regulated proteins showed that, at day 16 of initial culture and next to the cells divided once and the several-cell pro-embryos (expression phase of SE), there are still strong signals which can be
Conflict of interest
The authors declare no conflict of interest.
Author Contributions
LD performed 2-DE analysis, analyzed the data; AK-K and UJ performed the identification of proteins by mass spectrometry; MG and MS prepared the samples and carried out the microscopic analysis, LD, JJR and AM wrote the manuscript; AM conceived and designed the experiments. All authors read and approved the final manuscript.
Acknowledgments
The authors thank Ms. Ewa Znojek for perfect ultramicrotomy assistance. This research was supported by the Polish National Center for Science (NCN) research grant no. 2011/03/B/NZ9/02472.
References (103)
- et al.
Anatomical sequence and morphometric analysis during somatic embryogenesis on cultured cotyledon explants of Camellia japonica L
Ann Bot.
(1993) - et al.
Ultrastructural changes in cotyledons of pineapple guava (Myrtaceae) during somatic embryogenesis
Ann. Bot.
(1996) Ultrastructure of early secondary embryogenesis by multicellular and unicellular pathways in cork oak (Quercus suber L.)
Ann. Bot.
(2001)- et al.
Comparative quantitative proteomic analysis of embryogenic and non-embryogenic calli in maize suggests the role of oxylipins in plant totipotency
J. Proteomics.
(2014) - et al.
Proteome analysis of secondary somatic embryogenesis in cassava (Manihot esculenta)
Plant Sci.
(2008) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)- et al.
The sucrose-cleaving enzymes of plants are crucial for development, growth and carbon partitioning
Trends Plant Sci.
(1999) - et al.
Development of zygotic and somatic embryos of Phoenix dactylifera L. cv. Deglet Nour: Comparative study
Sci. Hortic. (Amsterdam).
(2008) - et al.
Abscisic acid and sucrose increase the protein content in date palm somatic embryos, causing changes in 2-DE profile
Phytochemistry
(2010) - et al.
Stress response proteins’ differential expression in embryogenic and non-embryogenic callus of Vitis vinifera L. cv. Cabernet Sauvignon-A proteomic approach
Plant Sci.
(2009)