Spatial expression of a sunflower SERK gene during induction of somatic embryogenesis and shoot organogenesis

Abstract

Organogenesis or somatic embryogenesis can be induced on immature zygotic embryos (IZE) of sunflower depending on the culture conditions. Both morphogenic processes originate from the same group of cells and show identical kinetics. Using real-time PCR and in situ hybridisation, we showed that somatic embryogenesis receptor-like kinase (SERK) transcripts accumulate early after the beginning of the culture in the morphogenic zone of IZE explants whatever the induction conditions used, i.e. organogenic, embryogenic or highly embryogenic conditions. Quantitative analyses failed to show any correlation between the SERK expression level during the period decisive for the orientation of the morphogenic pathway, i.e. the first 2 days of culture, and the type of morphogenesis induced. However, after 2 days of culture on the organogenic medium, the SERK gene expression level was severely down-regulated in the IZE explants. At 4 days of culture, SERK transcripts were no longer detectable by in situ hybridisation in the developing shoot structures whereas they still continued to accumulate in the embryonic structures induced on both embryogenic and highly embryogenic culture media. The significance of these expression analyses was addressed by transfer medium experiments. Results revealed that IZE cultured on the organogenic medium were able to form somatic embryos when transferred on the highly embryogenic medium as long as the SERK transcripts accumulated at a high level in their morphogenic zone, i.e. first 2 days of culture. Passt this delay, explants rapidly lost their embryogenic competence. Indeed, after 4 days of culture on the organogenic medium, IZE were definitely oriented towards shoot organogenesis. Taken together, these data suggest that reactive cells of IZE develop the competence to somatic embryogenesis during the first day of culture whatever the morphogenic induction conditions used.

Introduction

Somatic embryogenesis and shoot organogenesis are two different developmental pathways that both lead from somatic cells to entire new plants [23], [26]. Shoot organogenesis, also referred to as caulogenesis, is the process by which totipotent cells or tissues produce a unipolar structure, namely a shoot, whose vascular system is often connected to the parent tissues. In contrast, somatic embryogenesis leads to the production of a bipolar structure containing a root/shoot axis, with a closed independent vascular system [26]. During the initial phases of both organogenesis and embryogenesis processes, somatic cells must embark on a progression of developmental events referred to as dedifferentiation, competence acquisition, induction and determination [5], [15]. In spite of an accumulation of information about organogenesis and somatic embryogenesis, we still do not know why some in vitro cultured cells or tissues produce somatic embryos whereas others produce shoot primordia.

The in vitro cultured sunflower embryo system offers the unique opportunity to induce somatic embryogenesis or shoot organogenesis from the same population of cells and thus to conduct comparative studies on early events of both developmental pathways [3]. The morphogenic region or “reactive zone” is located at the upper border of the transition zone between hypocotyl and root tip of the immature zygotic embryos (IZE; Fig. 1B ), which corresponds to the future crown of the adult plant. The only growth regulator supplied in the medium necessary to initiate cell division is a cytokinin, the 6-benzyl amino purine (BAP). The morphogenic pathway can be oriented in a controllable manner using a set of precise culture conditions [4]. The timing of the morphogenic reaction induced is highly reproducible, fast, and identical in both somatic embryogenesis and organogenesis. The morphological and cytological changes occurring during the establishment of the two developmental processes have been previously described [3]. As early as 48 h after the beginning of the culture, the type of morphogenesis induced is already determined and can be identified on the basis of vacuole size, cytoplasm density and presence or absence of lipid droplets and protein bodies. However, such cytological traits are the visible consequences of an earlier initiation of specific genetic programs determining the cell identity which have still to be dissected. The identification and use of marker genes of the precocious initiation phases of either somatic embryogenesis or shoot organogenesis processes would be very helpful to further characterise the mechanisms underlying the orientation of the morphogenic pathway.

Plant receptor-like kinases (RLK) have been implicated in the cellular signalling of various developmental processes [2], [14]. The somatic embryogenesis receptor-like kinase or SERK gene encodes a leucine-rich repeat RLK and was found to mark single cells of carrot cell suspensions that are competent to regenerate through somatic embryogenesis [20]. Similar conclusions were drawn from expression studies on Dactylis glomerata and Arabidopsis thaliana orthologues of the carrot SERK gene [10], [22], suggesting an identical role for SERK in somatic embryogenesis among these plants. In contrast, the maize ZmSERK1 and ZmSERK2 genes are expressed in both embryogenic and non-embryogenic callus cultures [1]. However, Hecht et al. [10] show direct evidence that the AtSERK1 not only marks Arabidopsis cells competent to produce embryos but is also involved in the acquirement of the embryogenic competence. Indeed, plants overexpressing the AtSERK1 gene exhibited an enhanced capacity to initiate somatic embryogenesis during in vitro culture. This observation is in line with the up-regulation of AtSERK1 expression level existing in the altered meristem program one mutant [16] which possesses a natural high capacity to regenerate through somatic embryogenesis [10].

On the basis of these data, we hypothesised that the SERK-mediated pathway could trigger the orientation of the morphogenic response induced on sunflower IZE through somatic embryogenesis. On such a scenario, one would expect an up-regulation of SERK expression in the reactive cells of IZE cultured in embryogenic conditions. On the contrary, SERK expression would be repressed under organogenic conditions allowing the development of shoots. Another possibility is that the SERK-mediated pathway would be regulated at the level of, yet unknown, SERK activating-ligand(s) which would be only present and active under embryogenic conditions.

The aims of the work presented here were (a) to determine whether the SERK-mediated pathway is employed during the morphogenesis induced on cultured sunflower IZE and (b) to investigate whether there is a relationship existing between SERK expression level and the type of morphogenesis induced.

In situ hybridisation and real-time PCR were used to address the SERK expression pattern during the culture of IZE on three different media selected for their morphogenic potential, i.e. organogenesis, somatic embryogenesis and high somatic embryogenesis. In addition, medium transfer experiments were conducted to test how the competence to somatic embryogenesis is affected when IZE are cultured on an organogenic medium. Together these experimental approaches allowed us to improve our understanding about the mechanisms underlying the orientation of the morphogenic pathway taken by the reactive cells of in vitro cultured sunflower IZE.