Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells

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Abstract

Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

Introduction

A plant protoplast can be defined as a plant cell enclosed by the plasmalemma in the absence of the surrounding cell wall. Experimentally, plant protoplasts can be obtained from cells of various plant tissues by enzymatic cell wall removal. Cultured under appropriate conditions as single separated entities, protoplasts are capable of reforming their walls and dividing. That is why protoplast cultures are frequently used as a plant “free cell” system for experimental investigations (for reviews, [1], [2]). In addition to study basic cellular functions (cell division and elongation, cell wall synthesis, etc.), protoplast-derived cells can serve as models in developmental studies as well. Stress, exogenous hormones and the reactivation of cell division, all being associated with the isolation and culture of protoplasts, result in a flexible cellular state due to the general reorganization of chromatin structure, reprogramming of gene expression and cellular metabolism (reviewed by [1], [3]). This cellular flexibility favors a developmental switch in response to an appropriate developmental signal. Consequently, cell fate can be easily altered in protoplast-derived cell cultures and the underlying mechanisms can be approached at the cellular, molecular and biochemical levels.

For example, from alfalfa leaf protoplasts, embryogenic competent cells can be formed in response to 2,4-D [4], [5]. While a relatively low 2,4-D concentration (1 μM) results in the formation of elongated vacuolated cells, the embryogenic competent cell type can be observed in the presence of 10 μM 2,4-D. This latter type of cells could develop into proembryogenic cell clusters accumulating the AGL-15 protein in their nuclei as an embryogenic marker [4], [6]. It was therefore hypothesized, that the small, cytoplasmicaly dense protoplast-derived alfalfa cells represent a dedifferentiated cell state with the potency to initiate a new developmental program [3], [7].

In order to have a better insight into the cellular and molecular events associated with the formation of this embryogenic cell-type from alfalfa leaf protoplasts, a PCR-based cDNA subtraction approach was followed. This approach aimed the identification of transcripts up-regulated by 10 μM versus 1 μM 2,4-D in 4 days old leaf protoplast-derived cells. Relative expression of the identified 2,4-D up-regulated transcripts has been tested in other culture systems in order to verify their link to somatic embryogenesis.

Section snippets

Protoplast isolation and culture

Protoplasts were isolated from 4 to 6 weeks old alfalfa (Medicago sativa L. ssp. varia embryogenic genotype A2) plants grown in vitro on T1-medium [8] lacking phytohormones. Protoplast isolation and cultivation were done as described previously in details [4], [9].

RNA isolation and cDNA synthesis

In the case of protoplast cultures and leaf explants mRNA populations were isolated directly using the Dynabeads mRNA Direct Kit (Dynal Biotech, Oslo, Norway). Approximately the same number (200–250 × 105) of protoplast-derived cells or

cDNA subtraction-based identification of transcripts up-regulated by 10 μM 2,4-D

4-days-old leaf protoplast-derived alfalfa (Medicago sativa ssp. varia A2) cells from cultures with 1 μM or 10 μM 2,4-D, respectively, have been used for mRNA isolation and cDNA synthesis. Fig. 1A demonstrates the overall morphological difference between the cells grown in the presence of these 2,4-D concentrations. In addition to cell morphology, the gene expression pattern of the cells has also been specifically altered by the increased 2,4-D concentration, which is demonstrated by the

Discussion

Induction of somatic embryogenesis in protoplast-derived cells is a complex process that includes the recovery of cells from isolation stress, synthesis of a new cell wall, cell division and the acquisition of the embryogenic cell fate. It is supposed that increasing the 2,4-D concentration in the culture medium affected all of these parameters. This is supported by the identification of genes up regulated in response to the embryogenic 2,4-D concentration in the Medicago leaf

Acknowledgment

The presented experiments were partly supported by the Hungarian Scientific Research Fund (grants OTKA F34637 and T34818) and by CropDesign N.V. (Ghent, Belgium).

A. F. is thankful for the “János Bólyai” Research Fellowship for its support.

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    Present address: Institute of Biologie II, Ludwig-Albert University, Sonnerster 5., 79104 Freiburg, Germany.

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