The expression of LESK1 morphogenetic marker along the tomato hypocotyl axis is linked to a position-dependent competence for shoot regeneration

Abstract

Somatic morphogenesis is an important process whose mechanisms are still not completely understood. This is partially due to the lack of reliable morphogenetic markers that identify the different stages of the process. In the present work, a fragment of LESK1 gene (Lycopersicon esculentum shoot kinase 1) has been utilized as a marker of somatic caulogenesis. The expression of this marker pointed out a particular abundance of the corresponding transcript in tomato hypocotyls from 8-day-old plantlets. This elevated expression underlines the particular attitude to regeneration of this organ. Hypocotyl explants can in fact regenerate in the absence of exogenous growth regulators giving rise to shoot and root production at the apical and basal ends, respectively. The polarization observed in morphogenesis is always associated with polarized expression of LESK1 gene, which is elevated at the top of the explants, where shoot production occurs, and low at the basal pole, where roots were observed. A non-homogeneous LESK1 expression was also found along the hypocotyl axis where apparently marks cell populations with different competence to caulogenesis. These different levels of competence may be due to a variation in cell sensitivity to growth regulators, or the reaching of a well determined hormonal ratio required by the cells to be driven toward caulogenesis. LESK1 represents a reliable marker probably linked to the acquisition of competence for somatic caulogenesis.

Introduction

The mechanisms ruling somatic morphogenesis are conceivably very similar to those operating in the whole plant when, for instance, a traumatic event (such as wounding or cutting) induces the plant to regenerate new organs. The process takes place in cells that are already competent for morphogenesis (in function of the age or the developmental stage of the tissue they belong to) [1], or that acquire competence upon a triggering treatment such as the presence of particular nutrients [2], growth regulators [3], [4] or light [5]. After competence acquisition the cells become able to perceive the stimuli altering their developmental commitment, and are liable to induction in the presence of the appropriate stimulus that leads to organ determination [1], [3], [6], [7]. The subsequent phases involve cellular division, which leads to the formation of new meristems and to the development of new vascular structures, tissues, and organs, under the control of an efficient cell-to-cell communication. The complex network of biochemical and molecular pathways involved in the perception and transduction of all the regulatory signals mentioned, relies on the induction of specific genes whose activation is often spatially and transiently regulated and that can be considered markers of a determined phase of the developmental process. In recent years it has been found that many genes are involved in the acquisition of competence for cell division [8] or shoot induction [9], in the development of the vascular system [10], as well as in ruling and maintaining determined meristems [11], [12], [13], [14], [15].

The study and characterization of these genes could greatly improve the understanding of the molecular mechanisms underlying the different phases of in vitro morphogenesis and could consequently provide support in improving the effectiveness of this complex process (from the choice of explant source to the choice of culture conditions), as it is often tackled from a merely empirical point of view.

In a former study aimed at identifying the genes involved in somatic morphogenesis, from tomato cotyledons induced to produce shoots we isolated a cDNA clone (G36) 487 bp long [16] that exhibits the typical features of a morphogenetic marker. The expression of G36 gene is growth regulator dependent and is strongly enhanced in tomato cotyledons by means of the appropriate auxin/cytokinin ratio leading to somatic caulogenesis. In the present work, the screening of a cDNA library allowed us to isolate a longer cDNA clone containing the sequence of G36. This cDNA clone is highly homologous to many plant serine/threonine kinases, and thus it has been renamed LESK1 (Lycopersicon esculentum shoot kinase 1). In order to confirm the reliability of the LESK1 gene as molecular marker we have assessed its expression in various tissues with different regenerating capabilities. Our attention was focused on tomato hypocotyl. These explants, without supply of exogenous growth regulators, produce shoots and roots at the two ends showing a polarization in morphogenesis that fairly reproduces what occurs in the cuttings and represents a very suitable instrument for the study of in vitro morphogenesis.