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.

Cell layer-specific patterns of cell division and cell expansion during fruit set and fruit growth in tomato pericarp.

In angiosperms, the ovary wall resumes growth after pollination through a balanced combination of cell division and cell expansion. The quantitative pattern of these events remains poorly known in fleshy fruits such as tomato (Solanum spp.), in which dramatic growth of the pericarp occurs together with endoreduplication. Here, this pattern is reported at the level of each of the cell layers or groups of cell layers composing the pericarp, except for vascular bundles. Overall, cell division and cell expansion occurred at similar rates for 9 days post anthesis (DPA), with very specific patterns according to the layers. Subsequently, only cell expansion continued for up to 3-4 more weeks. New cell layers in the pericarp originated from periclinal cell divisions in the two sub-epidermal cell layers. The shortest doubling times for cell number and for cell volume were both detected early, at 4 DPA, in epicarp and mesocarp respectively, and were both found to be close to 14 h. Endoreduplication started before anthesis in pericarp and was stimulated at fruit set. It is proposed that cell division, endoreduplication, and cell expansion are triggered simultaneously in specific cell layers by the same signals issuing from pollination and fertilization, which contribute to the fastest relative fruit growth early after fruit set.

Fruit growth-related genes in tomato.

Tomato (Solanum lycopersicum Mill.) represents a model species for all fleshy fruits due to its biological cycle and the availability of numerous genetic and molecular resources. Its importance in human nutrition has made it one of the most valuable worldwide commodities. Tomato fruit size results from the combination of cell number and cell size, which are determined by both cell division and expansion. As fruit growth is mainly driven by cell expansion, cells from the (fleshy) pericarp tissue become highly polyploid according to the endoreduplication process, reaching a DNA content rarely encountered in other plant species (between 2C and 512C). Both cell division and cell expansion are under the control of complex interactions between hormone signalling and carbon partitioning, which establish crucial determinants of the quality of ripe fruit, such as the final size, weight, and shape, and organoleptic and nutritional traits. This review describes the genes known to contribute to fruit growth in tomato.

How fruit developmental biology makes use of flow cytometry approaches.

Fleshy fruit species such as tomato are important because of their nutritional and economic value. Several stages of fruit development such as ovary formation, fruit set, and fruit maturation have already been the subject of many developmental studies. However, fruit growth per se has been much less addressed. Fruit growth like all plant organs depends upon the developmental processes of cell division and cell expansion. The activity of cell divisions sets the number of cells that will compose the fruit; the cell expansion activity then determines its final size. Among the various mechanisms that may influence the determination of cell size, endopolyploidy by the means of endoreduplication, i.e. genome amplification in the absence of mitosis, appears to be of great importance in fleshy fruits. In tomato fruit, endoreduplication is associated with DNA-dependent cell expansion: cell size can reach spectacular levels such as hundreds of times its initial size (e.g. >0.5 mm in diameter), with as much as a 256-fold increase in nuclear DNA content. Using tomato fruit development as a model, recent investigations combining the use of flow cytometry, cellular imaging and molecular analyses have provided new data in favor of the long-standing karyoplasmic ratio theory, stating that cells tend to adjust their cytoplasmic volume to the nuclear DNA content. By establishing a highly structured cellular system where multiple physiological functions are integrated, endoreduplication acts as a morphogenetic factor supporting cell growth during tomato fruit development. In the context of plant breeding, deciphering the mechanisms controlling fruit growth, in particular those connecting the process of nuclear endoreduplication with modulation of gene expression, the regulation of cell size and final fruit size and composition, is necessary to understand better the establishment of fleshy fruit quality traits.

Endoreduplication and fruit growth in tomato: evidence in favour of the karyoplasmic ratio theory.

The growth of a plant organ depends upon the developmental processes of cell division and cell expansion. The activity of cell divisions sets the number of cells that will make up the organ; the cell expansion activity then determines its final size. Among the various mechanisms that may influence the determination of cell size, endopolyploidy by means of endoreduplication appears to be of great importance in plants. Endoreduplication is widespread in plants and supports the process of differentiation of cells and organs. Its functional role in plant cells is not fully understood, although it is commonly associated with ploidy-dependent cell expansion. During the development of tomato fruit, cells from the (fleshy) pericarp tissue become highly polyploid, reaching a DNA content barely encountered in other plant species (between 2C and 512C). Recent investigations using tomato fruit development as a model provided new data in favour of the long-standing karyoplasmic ratio theory, stating that cells tend to adjust their cytoplasmic volume to the nuclear DNA content. By establishing a highly structured cellular system where multiple physiological functions are integrated, endoreduplication does act as a morphogenetic factor supporting cell growth during tomato fruit development.

Evidence for karyoplasmic homeostasis during endoreduplication and a ploidy-dependent increase in gene transcription during tomato fruit growth.

Endopolyploidy is a widespread process that corresponds to the amplification of the genome in the absence of mitosis. In tomato, very high ploidy levels (up to 256C) are reached during fruit development, concomitant with very large cell sizes. Using cellular approaches (fluorescence and electron microscopy) we provide a structural analysis of endoreduplicated nuclei at the level of chromatin and nucleolar organisation, nuclear shape and relationship with other cellular organelles such as mitochondria. We demonstrate that endopolyploidy in pericarp leads to the formation of polytene chromosomes and markedly affects nuclear structure. Nuclei manifest a complex shape, with numerous deep grooves that are filled with mitochondria, affording a fairly constant ratio between nuclear surface and nuclear volume. We provide the first direct evidence that endopolyploidy plays a role in increased transcription of rRNA and mRNA on a per-nucleus basis. Overall, our results provide quantitative evidence in favour of the karyoplasmic theory and show that endoreduplication is associated with complex cellular organisation during tomato fruit development.

In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH).

Endopolyploidy, i.e. amplification of the genome in the absence of mitosis, occurs in many plant species and happens along with organ and cell differentiation. Deciphering the functional roles of endopolyploidy is hampered by the fact that polyploid tissues generally comprise cells with various ploidy levels. In some fleshy fruits (amongst them tomato fruit) the ploidy levels present at the end of development range from 2C to 256C in the same tissue. To investigate the temporal and spatial distribution of endopolyploidy it is necessary to address the DNA content of individual nuclei in situ. Conventional methods such as fluorometry or densitometry can be used for some tissues displaying favorable characteristics, e.g. small cells, small nuclei, organization in a monolayer, but high levels of varying polyploidy are usually associated with large sizes of nuclei and cells, in a complex three dimensional (3-D) organization of the tissues. The conventional methods are inadequate for such tissue, becoming semi-quantitative and imprecise. We report here the development of a new method based on fluorescent in situ bacterial artificial chromosome hybridizations that allows the in situ determination of the DNA ploidy level of individual nuclei. This method relies on the counting of hybridization signals and not on intensity measurements and is expected to provide an alternative method for mapping endopolyploidy patterns in mature, 3-D organized plant tissues as illustrated by the analysis of ploidy level and cell size in pericarp from mature green tomato fruit.

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.

Functional characterization of the HD-ZIP IV transcription factor OCL1 from maize.

OCL1 (OUTER CELL LAYER1) encodes a maize HD-ZIP class IV transcription factor (TF) characterized by the presence of a homeo DNA-binding domain (HD), a dimerization leucine zipper domain (ZIP), and a steroidogenic acute regulatory protein (StAR)-related lipid transfer domain (START) involved in lipid transport in animals but the function of which is still unknown in plants. By combining yeast and plant trans-activation assays, the transcriptional activation domain of OCL1 was localized to 85 amino acids in the N-terminal part of the START domain. Full-length OCL1 devoid of this activation domain is unable to trans-activate a reporter gene under the control of a minimal promoter fused to six repeats of the L1 box, a cis-element present in target genes of HD-ZIP IV TFs in Arabidopsis. In addition, ectopic expression of OCL1 leads to pleiotropic phenotypic aberrations in transgenic maize plants, the most conspicuous one being a strong delay in flowering time which is correlated with the misexpression of molecular markers for floral transition such as ZMM4 (Zea Mays MADS-box4) or DLF1 (DELAYED FLOWERING1). As suggested by the interaction in planta between OCL1 and SWI3C1, a bona fide subunit of the SWI/SNF complex, OCL1 may modulate transcriptional activity of its target genes by interaction with a chromatin remodelling complex.

Functional characterization of the tomato cyclin-dependent kinase inhibitor SlKRP1 domains involved in protein-protein interactions.

• Cyclin-dependent kinase (CDK) inhibitors (kip-related proteins, KRPs) play a major role in the regulation of plant cell cycle in antagonizing its progression, and are thus regulators of development. The primary sequence of KRPs is characterized by the existence of conserved motifs, for which we have limited information on their functional significance. • We performed a functional analysis of various domains present in KRPs from tomato. A series of deletion mutants of SlKRP1 was generated and used in transient expression assays to define the relevance of conserved protein domains in subcellular and subnuclear localizations. Specific interactions of SlKRP1 and its deletion variants with cell cycle proteins were investigated using two-hybrid assays and bimolecular fluorescent complementation. • Plant KRPs are distributed into two phylogenetic subgroups according to the presence of conserved motifs. Members of subgroup 1 represented by SlKRP1 share 6 conserved motifs whose function in protein localization and protein-protein interactions could be identified. A new interaction motif was localized in the central part of SlKRP1 that targets SlCDKA1 and SlCYCD3;1 to the nucleus. • Our results bring new insights to the functional role of particular domains in KRPs relative to subcellular localization or proteolytic degradation.

Cell expansion and endoreduplication show a large genetic variability in pericarp and contribute strongly to tomato fruit growth.

Postanthesis growth of tomato (Solanum lycopersicon) as of many types of fruit relies on cell division and cell expansion, so that some of the largest cells to be found in plants occur in fleshy fruit. Endoreduplication is known to occur in such materials, which suggests its involvement in cell expansion, although no data have demonstrated this hypothesis as yet. We have analyzed pattern formation, cell size, and ploidy in tomato fruit pericarp. A first set of data was collected in one cherry tomato line throughout fruit development. A second set of data was obtained from 20 tomato lines displaying a large weight range in fruit, which were compared as ovaries at anthesis and as fully grown fruit at breaker stage. A remarkable conservation of pericarp pattern, including cell layer number and cell size, is observed in all of the 20 tomato lines at anthesis, whereas large variations of growth occur afterward. A strong, positive correlation, combining development and genetic diversity, is demonstrated between mean cell size and ploidy, which holds for mean cell diameters from 10 to 350 microm (i.e. a 32,000-times volume variation) and for mean ploidy levels from 3 to 80 C. Fruit weight appears also significantly correlated with cell size and ploidy. These data provide a framework of pericarp patterning and growth. They strongly suggest the quantitative importance of polyploidy-associated cell expansion as a determinant of fruit weight in tomato.

Differential sensitivity of plant and yeast MRP (ABCC)-mediated organic anion transport processes towards sulfonylureas.

The role of ATP-binding cassette (ABC) proteins such as multidrug resistance-associated proteins (MRPs) is critical in drug resistance in cancer cells and in plant detoxification processes. Due to broad substrate spectra, specific modulators of these proteins are still lacking. Sulfonylureas such as glibenclamide are used to treat non-insulin-dependent diabetes since they bind to the sulfonylurea receptor. Glibenclamide also inhibits the cystic fibrosis transmembrane conductance regulator, p-glycoprotein in animals and guard cell ion channels in plants. To investigate whether this compound is a more general blocker of ABC transporters the sensitivity of ABC-type transport processes across the vacuolar membrane of plants and yeast towards glibenclamide was evaluated. Glibenclamide inhibits the ATP-dependent uptake of beta-estradiol 17-(beta-D-glucuronide), lucifer yellow CH, and (2',7'-bis-(2-carboxyethyl)-5-(and-6-)carboxyfluorescein. Transport of glutathione conjugates into plant but not into yeast vacuoles was drastically reduced by glibenclamide. Thus, irrespective of the homologies between plant, yeast and animal MRP transporters, specific features of plant vacuolar MRPs with regard to sensitivity towards sulfonylureas exist. Glibenclamide could be a useful tool to trap anionic fluorescent indicator dyes in the cytosol.

TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19.

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrug-resistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.

ZmEBE genes show a novel, continuous expression pattern in the central cell before fertilization and in specific domains of the resulting endosperm after fertilization.

Two novel maize genes expressed specifically in the central cell of the female gametophyte and in two compartments of the endosperm (the basal endosperm transfer layer and the embryo surrounding region) were characterized. The ZmEBE (embryo sac/basal endosperm transfer layer/embryo surrounding region) genes were isolated by a differential display between the upper and the lower half of the kernel at 7 days after pollination (DAP). Sequence analysis revealed ORFs coding for two closely related proteins of 304 amino acids (ZmEBE-1) and 286 amino acids (ZmEBE-2). This size difference was due to differences in the splicing of the two genes. Both protein sequences showed significant similarity to the DUF239 family of Arabidopsis, a group of 22 proteins of unknown function, a small number of which are putative peptidases. ZmEBE genes had a novel cell type-specific expression pattern in the central cell before and the resulting endosperm after fertilization. RT-PCR analysis showed that the expression of both genes started before pollination in the central cell and continued in the kernel up to 20 DAP with a peak at 7 DAP. In situ hybridization revealed that the expression in the kernel was restricted to the basal transfer cell layer and the embryo surrounding region of the endosperm. The expression of ZmEBE-1 was at least 10 times lower than that of ZmEBE-2. Similarly to other genes expressed in the endosperm, ZmEBE-1 expression was subject to a parent-of-origin effect, while no such effect was detected in ZmEBE-2. Sequence analysis of upstream regions revealed a potential cis element of 33 bp repeated 7 times in ZmEBE-1 and ZmEBE-2 between positions -900 and -100. The 1.6 kb ZmEBE-2 upstream sequence containing the seven R7 elements was able to confer expression in the basal endosperm to a Gus reporter gene. These data indicate that ZmEBE is potentially involved in the early development of specialized domains of the endosperm and that this process is possibly already initiated in the central cell, which is at the origin of the endosperm.

The destination for single-pass membrane proteins is influenced markedly by the length of the hydrophobic domain.

The tonoplast was proposed as a default destination of membrane-bound proteins without specific targeting signals. To investigate the nature of this targeting, we created type I fusion proteins with green fluorescent protein followed by the transmembrane domain of the human lysosomal protein LAMP1. We varied the length of the transmembrane domain from 23 to either 20 or 17 amino acids by deletion within the hydrophobic domain. The resulting chimeras, called TM23, TM20, and TM17, were expressed either transiently or stably in tobacco. TM23 clearly accumulated in the plasmalemma, as confirmed by immunoelectron microscopy. In contrast, TM17 clearly was retained in the endoplasmic reticulum, and TM20 accumulated in small mobile structures. The nature of the TM20-labeled compartments was investigated by coexpression with a marker localized mainly in the Golgi apparatus, AtERD2, fused to a yellow fluorescent protein. The strict colocalization of both fluorescent proteins indicated that TM20 accumulated in the Golgi apparatus. To further test the default destination of type I membrane proteins, green fluorescent protein was fused to the 19-amino acid transmembrane domain of the plant vacuolar sorting receptor BP-80. The resulting chimera also accumulated in the Golgi instead of in post-Golgi compartments, where native BP-80 localized. Additionally, when the transmembrane domain of BP-80 was lengthened to 22 amino acids, the reporter escaped the Golgi and accumulated in the plasma membrane. Thus, the tonoplast apparently is not a favored default destination for type I membrane proteins in plants. Moreover, the target membrane where the chimera concentrates is not unique and depends at least in part on the length of the membrane-spanning domain.

Flavone glucoside uptake into barley mesophyll and Arabidopsis cell culture vacuoles. Energization occurs by H(+)-antiport and ATP-binding cassette-type mechanisms.

In many cases, secondary plant products accumulate in the large central vacuole of plant cells. However, the mechanisms involved in the transport of secondary compounds are only poorly understood. Here, we demonstrate that the transport mechanisms for the major barley (Hordeum vulgare) flavonoid saponarin (apigenin 6-C-glucosyl-7-O-glucoside) are different in various plant species: Uptake into barley vacuoles occurs via a proton antiport and is competitively inhibited by isovitexin (apigenin 6-C-glucoside), suggesting that both flavone glucosides are recognized by the same transporter. In contrast, the transport into vacuoles from Arabidopsis, which does not synthesize flavone glucosides, displays typical characteristics of ATP-binding cassette transporters. Transport of saponarin into vacuoles of both the species is saturable with a K(m) of 50 to 100 microM. Furthermore, the uptake of saponarin into vacuoles from a barley mutant exhibiting a strongly reduced flavone glucoside biosynthesis is drastically decreased when compared with the parent variety. Thus, the barley vacuolar flavone glucoside/H(+) antiporter could be modulated by the availability of the substrate. We propose that different vacuolar transporters may be responsible for the sequestration of species-specific/endogenous and nonspecific/xenobiotic secondary compounds in planta.

Between myth and reality: genetically modified maize, an example of a sizeable scientific controversy.

Maize is a major crop plant with essential agronomical interests and a model plant for genetic studies. With the development of plant genetic engineering technology, many transgenic strains of this monocotyledonous plant have been produced over the past decade. In particular, field-cultivated insect-resistant Bt-maize hybrids are at the centre of an intense debate between scientists and organizations recalcitrant to genetically modified organisms (GMOs). This debate, which addresses both safety and ethical aspects, has raised questions about the impact of genetically modified (GM) crops on the biodiversity of traditional landraces and on the environment. Here, we review some of the key points of maize genetic history as well as the methods used to stably transform this cereal. We describe the genetically engineered Bt-maizes available for field cultivation and we investigate the controversial reports on their impacts on non-target insects such as the monarch butterfly and on the flow of transgenes into Mexican maize landraces.