CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria

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:evidences in favour of the karyoplasmic ratio theory

Endopolyploidy occurs in many plant species and supports the process of differentiation of cells and organs. The functional role of endopolyploidy in plant cells remains poorly understood, mainly because the analysis is hampered by the fact that complex polyploid tissues usually include cells with different ploidy levels. During the development of tomato fruit, cells from the (fleshy) pericarp tissue become highly polyploid reaching DNA content barely encountered in other plant species (between 2C and 512C). To investigate the spatial and temporal distribution of endopolyploidy, it is necessary to address the DNA content of individual nuclei in situ. Populations of nuclei with different ploidy levels were isolated to characterize at the cytological level the consequences of endopolyploidy on the ultrastructure of nuclear and nucleolar chromatin, the nuclear shape and the relationship with other cellular organelles such as mitochondria. We were able to develop a new method based on BAC-FISH to determine in situ the ploidy level of different nuclei and consequently establish a ploidy map of tomato fruit pericarp. Based on this map, we demonstrated a link between the ploidy level, the complexity of nuclear shape and the number of mitochondria in the vicinity of polyploid nuclei. We were able to provide the first direct evidence that endoreduplication plays a role in the increased transcription of rRNA and mRNA in plant cells. We thus provided quantitative data in favour of the ‘karyoplasmic ratio’ theory and showed that endoreduplication is associated with a complex cellular re-organization during development of tomato fruit

The anaphase promoting complex activator CCS52A, a key factor for fruit growth and endoreduplication in tomato

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endoreduplication in connection to cell expansion and final fruit size determination. Endoreduplication occurs as an impairment of mitosis, which can originate from the selective degradation of M-phase-specific cyclins via the ubiquitin-mediated proteolytic pathway, requiring the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C). In plants CCS52A is the ortholog of CDH1/FZR proteins from yeast, drosophila and human, belonging to the WD40-repeat protein family. During fruit development, the SlCCS52A gene expression is specifically associated to endoreduplication in tomato. Altering SlCCS52A expression in either negative or positive manner impacts the extent of endoreduplication in fruit and affects fruit size. When SlCCS52A is down-expressed endoreduplication is impaired during fruit growth leading to reduced fruit growth. However when SlCCS52A is overexpressed, endoreduplication is initially delayed, accounting for the altered final fruit size, but resumes and is even enhanced leading to fruit growth recovery, pointing at the physiological role of endoreduplication in growth induction during tomato fruit development