Jean-Pierre Renaudin

Elucidating the functional role of endoreduplication in tomato fruit development.

BACKGROUND Endoreduplication is the major source of endopolyploidy in higher plants. The process of endoreduplication results from the ability of cells to modify their classical cell cycle into a partial cell cycle where DNA synthesis occurs independently from mitosis. Despite the ubiquitous occurrence of the phenomenon in eukaryotic cells, the physiological meaning of endoreduplication remains vague, although several roles during plant development have been proposed, mostly related to cell differentiation and cell size determination. SCOPE Here recent advances in the knowledge of endoreduplication and fruit organogenesis are reviewed, focusing on tomato (Solanum lycopersicum) as a model, and the functional analyses of endoreduplication-associated regulatory genes in tomato fruit are described. CONCLUSIONS The cyclin-dependent kinase inhibitory kinase WEE1 and the anaphase promoting complex activator CCS52A both participate in the control of cell size and the endoreduplication process driving cell expansion during early fruit development in tomato. Moreover the fruit-specific functional analysis of the tomato CDK inhibitor KRP1 reveals that cell size and fruit size determination can be uncoupled from DNA ploidy levels, indicating that endoreduplication acts rather as a limiting factor for cell growth. The overall functional data contribute to unravelling the physiological role of endoreduplication in growth induction of fleshy fruits.

A thiol protease and an anionic peroxidase are induced by lowering cytokinins during callus growth in Petunia

We previously identified a group of proteins that increase early in Petunia hybrida calli subcultured on a low-cytokinin medium, unlike the calli subcultured on a high-cytokinin medium. The calli on the low-cytokinin medium do not regenerate (J.-P. Renaudin, C. Tournaire, B. Teyssendier de la Serve [1991] Physiol Plant 82: 48–56). Two of these proteins, P21 and P17, have been identified by peptide sequencing and cloned. P21 is highly homologous to a group of thiol proteases, including barley aleurain, rice oryzain [gamma], Arabidopsis SAG2, and mammalian cathepsin H. P17 is highly homologous to a group of anionic peroxidases from potato and tomato. A study of their expression in two P. hybrida lines, PC6 and St40, which differ in their ability to regenerate, showed that the genes for P21 and P17 are differentially expressed depending on the type and the age of the organ, with the highest expression in senescing leaves and in aged calli. The data are in favor of these genes being associated with an early step of senescence, which may be due, in part, to a reduction in total cytokinin. The two Petunia lines are, thus, functionally different concerning the action of cytokinin in two developmental phenomena: in vitro organogenesis and senescence.

Cytokinins modulate the steady-state levels of light-dependent and light-independent proteins and mRNAs in tobacco cell suspensions

Abstract Cytokinin modulation of chloroplast differentiation in light-grown tobacco ( Nicotiana tabacum cv Wisconsin 38) cell suspensions was previously shown to involve the control of specific nuclear-encoded plastid proteins, including light-harvesting complex chlorophyll a / b binding protein (Cab) and the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (SSU). The steady-state levels of the corresponding mRNAs was enhanced in response to the hormone. Patterns of gene expression in response to cytokinin and light were further compared. Cytokinin-deprived, light-grown tobacco cells were subcultured, either in the dark or under white light, in a kinetin-supplemented medium or in a cytokinin-free medium. Total cell proteins were separated by two-dimensional electrophoresis. Amongst more than 500 distinguishable silver stained polypeptide spots, only about twenty were found to vary as a function of culture time and of light or cytokinin suppl. These variations were quantified by image analysis. Various interactions between light and cytokinin in modulating the steady-state levels of some polypeptides were observed. Independently of light, cytokinin increased or decreased the levels of some polypeptides. These polypeptides were different from pathogenesis-related polypeptides detected in virus infected tobacco leaves. In vitro translation products of poly(A) + RNAs purified from cells harvested during the growth phase were fractionated by two-dimensional electrophoresis: some translatable mRNAs were modulated by kinetic but not by light. Northern blot and slot-blot hybridization experiments showed that kinetin enhanced the steady-levels of Cab and SSU encoding mRNAs in light-grown cells only and had no detectable effect on these mRNAs in the dark.

Changes in microtubular organization mark the transition to organized growth during organogenesis in Petunia hybrida

Abstract We have studied microtubular organization during the induction of organogenesis in callus derived from leaf protoplasts of Petunia hybrida , line PC6. The conditions for regeneration have been determined in detail and it is possible to obtain and compare calli which differ in their competence to regenerate. The main conclusions are: (1) that dividing cells in non-polarized callus have a low relative amount of preprophase bands (PPB-index), regardless of their potential to regenerate; (2) that this low PPB-index is due to the absence of PPBs in preprophase and early prophase; (3) that a 3–5-fold increase in the PPB-index marks the transition to organized growth; (4) that the interphase cytoskeleton is unordered in the non-organized callus and that ordered, helical arrays of interphase microtubules do not appear before polarized division has been restored.

Sequential hormone requirement for growth and organogenesis of Petunia hybrida protoplasts-derived calli

Abstract A procedure to regenerate buds or roots from little calli derived from Petunia hybrida leaf protoplasts has been developed. It comprises three culture periods on simple, wholly defined media. Protoplasts were first plated at high density for 7 days to induce initial divisions. Microcolonies were then plated at low density and grown iittle calli for 10–30 days, either in Petri dishes or in large volume cultures. During this step, auxin and cytokinin concentrations, and interactions between these hormones, influenced markedly the plating efficiency of microcolonies and the capacity of calli to sustain transfer to low osmotic strength media. All calli regenerated buds within 20 days after transfer onto a cytokinin-rich solid medium, in which osmoticum, sugar and cytokinin concentrations have been optimized. Auxin requirement depended on auxin and cytokinin concentrations supplied during the previous growth step. When the calli were transferred onto an auxin-rich solid medium, roots formed mostly from those which had previously grown in the presence of large cytokinin concentrations. These sequential hormone effects on growth and regeneration make this system suitable for further analysis of the physiological determination of competence and for a molecular approach of hormone action on organogenesis.

Ajmalicine Transport into Vacuoles Isolated from Catharanthus Roseus Cells

We have studied previously the compartmentation of ajmalicine in Catharanthus roseus cells (Renaudin and Guern, 1982; Renaudin et al., 1985) and of nicotine in Nicotiana tabacum and Acer pseudoplatanus cells (Kurkdjian, 1982). The behavior of the 14C-labelled alkaloids added in the cell suspensions was in good agreement with the predictions of the so-called ion-trapping model: These alkaloids apparently diffused passively through the plasmalemma and the tonoplast, mainly under their neutral form, and did accordingly distribute between the cells and the medium. They were accumulated within cells, likely in vacuoles, against a concentration gradient, because of the relative acidity of the vacuolar compartment and of the low capacity for diffusion of the alkaloid cation. The distribution of alkaloids between the cells (i.e. the vacuoles) and the medium was dynamic and it was a function of 1) the acidity constant of the alkaloid, and 2) the pH difference between the vacuole and the medium. Due to the high pH (ca. 7.5) and low relative volume (less than 20%) of the cytoplasm, the accumulation of this compartment was likely neglectable. It was assumed that accumulation in whole cells merely reflected the accumulation within vacuoles, thus allowing the indirect approach of vacuolar properties.

Properties of Vacuoles as a Function of the Isolation Procedure

A more and more detailed picture of the properties of vacuoles is emerging from the literature of the past few years. The vacuoles are major storage compartments for acid hydrolases, sucrose, organic acids such as malate and citrate, basic amino-acids and many different secondary metabolites. Aside this general and coherent picture, large discrepancies exist in the literature concerning the intensity of the electrochemical potential difference of protons (Δ\(\overline{\mu {{H}^{+}}}\)) across the tonoplast of isolated vacuoles. The electrical potential difference (Em) across the tonoplast of isolated vacuoles was reported to be negative when calculated from the equilibrium distribution of permeant lipophilic cations, whereas positive Em were measured with microelectrodes (for a review, see Leigh, 1983, and Gibrat et al., 1985 a). As to the transtonoplast pH gradient, the only agreement is on the fact that the vacuoles are acidic relative to the cytoplasm. But very few measurements of the transtonoplast ΔpH in cells or isolated vacuoles have been performed. Furthermore, reports of the dissipation of this pH gradient during the isolation of vacuoles and their subsequent manipulation (Schmitt and Sandermann, 1982; Matern et al., 1986) contrast with other results demonstrating the stability of the ΔpH.

Accumulation of Organic Solutes in Plant Vacuoles: The Interpretation of Data is Not so Easy

Significant progress has been made recently about the mechanisms of transport of solutes across the tonoplast (Alibert and Boudet, 1982; Leigh, 1983; Deus-Neumann and Zenk, 1984; Boller, 1985; Thom and Maretzki, 1985; Deus-Neumann and Zenk, 1986; Thom et al., 1986). The diversity of transport systems tentatively identified at the tonoplast appears rather large (Guern et al., 1987). A few examples illustrate the various types of transmembrane transfer involved in the vacuolar exchanges.

Growth and Physiology of Suspension-Cultured Plant Cells: the Contribution of Tobacco BY-2 Cells to the Study of Auxin Action

Cultured animal and plant cells display unique advantages such as the homogeneity of cell types within a population, the control of extracellular parameters and the continuous availability of biomass. In addition, the observation and manipulation of cultured cells is easier than that of cells within the organs. Cultured animal cells have been profitably used for the analysis of various differentiation processes. The differentiated status of plant cells is more reversible than that of animal cells, as it is in a major part the consequence of the numerous signals that they perceive in planta or in vitro. Plant cells cultured in vitro are often said to be undifferentiated. In fact, they do not resemble truly undifferentiated plant cells such as meristematic ones, as they share signs of dedifferentiation like proplastids and mitotic activity, with signs of differentiation like large vacuolar and Golgi apparatus and a relatively thick wall. In addition, in vitro cultured plant cells are much larger than meristematic cells. Cultured plant cells are widely used as convenient biomass providing systems that undergo basic mechanisms such as cell division. Their suitability for the approach to whole plant biology has been repeatedly questioned because of the peculiar differentiation status they adopt in vitro. However, their capacity to provide models of differentiation has already been demonstrated, e.g., in somatic embryogenesis, meristem neoformation, xylogenesis, photosynthesis, and secondary metabolites biosynthesis. Moreover, cultured plant cells do react to hormones and other signals as well as cells in planta. They provide ideal tools to study the cellular and molecular relationship of the effect of the signals associated with totipotency in plant cells.

Transcriptome profiling of sorted endoreduplicated nuclei from tomato fruits: how the global shift in expression ascribed to DNA ploidy influences RNA-Seq data normalization and interpretation

As part of normal development most eukaryotic organisms, ranging from insects and mammals to plants, display variations in nuclear ploidy levels resulting from somatic endopolyploidy. Endoreduplication is the major source of endopolyploidy in higher plants. Endoreduplication is a remarkable characteristic of the fleshy pericarp tissue of developing tomato fruits, where it establishes a highly integrated cellular system that acts as a morphogenetic factor supporting cell growth. However, the functional significance of endoreduplication is not fully understood. Although endoreduplication is thought to increase metabolic activity due to a global increase in transcription, the issue of gene-specific ploidy-regulated transcription remains open. To investigate the influence of endoreduplication on transcription in tomato fruit, we tested the feasibility of a RNA sequencing (RNA-Seq) approach using total nuclear RNA extracted from purified populations of flow cytometry-sorted nuclei based on their DNA content. Here we show that cell-based approaches to the study of RNA-Seq profiles need to take into account the putative global shift in expression between samples for correct analysis and interpretation of the data. From ploidy-specific expression profiles we found that the activity of cells inside the pericarp is related both to the ploidy level and their tissue location.