Functional analysis of the bell pepper KNOLLE gene (cakn) promoter region in tobacco plants and in synchronized BY2 cells

Abstract

Cytokinesis in higher plants, the final stage of cell division, is accomplished by the formation of the cell plate, a unique cytokinetic membranous organelle that is assembled across the inside of the dividing cell. There is evidence for a major role played by secretory vesicle trafficking and fusion in this process and the KNOLLE protein, specifically involved in vesicle fusion during the building of the cell plate, is one of the best-characterized component. Previously we had determined the expression of the bell pepper KNOLLE gene (cakn) in dividing tissues. In the present study, we demonstrate that 1372 bp of its promoter region are able to drive the expression of the GUS (beta-glucuronidase) reporter gene in tobacco tissues containing highly dividing cells. In synchronized tobacco BY2 cells, a 261 bp fragment of the promoter confers a G2 and M-phase-specific expression on the GUS reporter gene. This DNA fragment contains two Myb-like cis-elements identical to the MSA elements characterized in B-type cyclin promoters. Our results suggest the existence of equivalent regulation mechanisms for a gene encoding a protein involved in the building of the cell plate, KNOLLE, and genes coding for proteins associated with the regulation of the cell cycle such as cyclins.

Introduction

Coordinated cell divisions and differentiation mediate the development of a whole plant from a single cell, suggesting an effect of the activity of key cell cycle regulators on this process. But as propounded, the cell cycle is neither slave nor master of the development but is integrated into the complex pathways of morphogenesis and histogenesis [1]. In plants, as in other eukaryotes, the cell cycle involves temporal succession of phases of DNA replication (S-phase) and mitosis (M-phase), which are separated by gap stages (G) in a sequence G1–S–G2–M. The cyclin-dependent kinases (CDKs) and their cyclin partners are major control regulators of the cell cycle. The regulatory processes involved in the transitions between the different steps are at the same time transcriptional (modulations of the transcript level) and post-transcriptional (variations of protein abundance and/or protein activity). The G2/M transition is triggered by a CDK activity after the association of the kinase with a cyclin B and the phase-dependent expression of B-type cyclins is under transcriptional control [2]. Several B-type cyclin gene promoters contain a common cis-acting element, the mitotic specific activator (MSA) that is necessary and sufficient for M-phase-specific expression [3], [4]. This motif, closely matching the consensus sequences of c- and v-Myb binding sites, is also present in the tobacco kinesin-like motor protein gene NACK1 which shows an expression profile similar to that of B-type cyclin genes [4]. The NACK1 protein, a microtubule-associated protein, acts as activator of the tobacco mitogen-activated protein kinase NPK1 [5]. This results suggest that a number of G2 and/or M-phase-specific genes could be coordinately expressed through a common MSA-mediated mechanism.

Cell division ends with cytokinesis, leading to the appropriate distribution of nuclear and cytoplasmic genetic information to daughter cells. In higher plants, rather than constricting the periphery as in many bacteria and in animals, cells build their partition outwards by using a specialized structure called the cell plate. This intriguing intracellular compartment arises de novo by targeted transport and fusion of vesicles, and then expands centrifugally to form a disc that aggregates and fuses with the plasma membrane at a position determined prior to the onset of the mitosis [6]. Vesicle transport/fusion events are critical for the formation of the new cell wall. A number of proteins were found to be involved in the formation of the cell plate, among them proteins associated with the vesicle fusion complex like the syntaxin KNOLLE [7], [8], [9] and its associated proteins SNAP33, NSPN11, KEULE and AtCDC48 ([10] and references herein (Akrim and Houlné unpublished results). KNOLLE is a member of the soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNARE) protein family, involved in membrane fusion during intracellular membrane traffic [11]. It is specifically detected at the cell-plate level in dividing tissues and rapidly disappears at the completion of cytokinesis [7], [8], [9]. However, studies on such proteins mainly concern their function and localization and much less the expression of the corresponding genes.

The aim of our work is to unravel the mechanisms involved in the early stages of fruit development involving cell division processes using the fleshy fruit of bell pepper (Capsicum annuum) as a model. Early fruit development phase is one of the few stages of development during which cell division is swiftly and highly activated in response to one or several specific signals [12]. Dissecting the promoter region of KNOLLE gene will allow a better understanding of the mechanisms controlling the transcriptional regulation of this gene and help to decipher the signalization pathway integrating the cell plate formation in the cell cycle.

In this study, we describe the characterization and the analysis of the promoter region of the bell pepper KNOLLE gene cakn. We show that this promoter sequence drives the expression of the GUS reporter gene in tobacco dividing tissues as well as in tobacco BY2 cells. In synchronized tobacco BY2 cells we show that a −267 fragment of the cakn promoter is able to drive the expression of the reporter gene with the same late G2 and M specificity as ntkn, the tobacco gene orthologous of cakn. The putative roles of two Myb-like cis-elements identical to those previously identified in B-type cyclin promoters is discussed. This is the first analysis of the promoter of a gene encoding a protein structurally-associated with the formation of the cell plate. It should allow to define a relationship at the level of regulatory mechanisms, between the expression of the cakn gene and genes encoding proteins controlling the progress of the cell cycle during G2- and M-phase such as cyclins.