Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato

Abstract

SUMMARYTomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo-reduplication in connection with cell expansion and final fruit size determination. Endo-reduplication is an impairment of mitosis that originates 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). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo-reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo-reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over-expressing fruits, endo-reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo-reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo-reduplication in growth induction during tomato fruit development.

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

“…Although the positive correlation between endoreduplication and cell size has been described in wild-type tomato fruits Nafati et al, 2011), we have shown that there is a broad variety of cell types from IE to OE according to a complex differentiation pattern during fruit development, including a large discrepancy in cell sizes with partially overlapping ploidy levels . These data and additional functional studies led us to propose that endoreduplication serves as a trigger for organ growth and provides the potential to support a range of cell sizes rather than any precise, definitive size (Mathieu-Rivet et al, 2010;Nafati et al, 2011). The nuclear morphology of endoreduplicated cells in tomato pericarp is strongly affected by endopolyploidisation, with highly invaginated and grooved nuclear envelopes (Fig.…”

“…Previous functional analyses of genes regulating the endocycle (Gonzalez et al, 2007;Mathieu-Rivet et al, 2010;Nafati et al, 2011) demonstrated the biological significance of endoreduplication as a morphogenetic factor for cell and fruit growth in tomato. In addition, enhancing endoreduplication and consequently fruit growth also strongly impacted nitrogen metabolism in developing fruits (Mathieu-Rivet et al, 2010), establishing a link between endoreduplication-induced growth and primary metabolism, as reported in maize endosperm (Leiva-Neto et al, 2004).…”

“…To address gene expression during endoreduplication, we selected five single-copy protein-coding genes in tomato: RPB1, which encodes the large subunit of RNA pol II; the endocycle regulating genes CCS52A and WEE1 (Gonzalez et al, 2007;Mathieu-Rivet et al, 2010); and two mitosis-associated genes used as negative controls of gene expression in endoreduplicated nuclei, encoding CDKB2, a cyclin-dependent kinase specific to the G2-M transition (Chevalier, 2007), and the kinetochore MIS12 protein (Sato et al, 2005). qPCR on genomic DNA extracted from 4C, 16C and 32C sorted nuclei confirmed that the extent of all five gene duplications was linearly correlated with the degree of nuclear polyploidisation (supplementary material Fig.…”

The structural and molecular analysis of endoreduplicated nuclei in tomato (Solanum lycopersicum) fruit provides evidence for a ploidy-dependent increase in transcriptional activity

“…We selected five single-copy genes coding for the following proteins from tomato: RPB1, the large subunit of RNA pol II; CCS52A and WEE1, two key proteins involved in the regulation of the endocycle (Gonzalez et al 2007;Mathieu-Rivet et al 2010); two mitosis-associated proteins: CDKB2, a cyclin-dependent kinase specific to the G2-M transition (Chevalier 2007) and the kinetochore MIS12 protein (Sato et al 2005). The genes encoding these two mitosis-specific proteins were thus used as negative controls of gene expression in endoreduplicated nuclei.…”

“…Taking advantage of the extraordinary extent of the endoreduplication process occurring in tomato fruit, we contributed to provide new and important insights on the molecular mechanisms triggering the exit of the mitotic cycle towards the onset of the endoreduplication cycle associated to cell expansion (for a review, see Chevalier et al 2011). We demonstrated very clearly that modifying endoreduplication during fruit development impacts on fruit growth and final fruit size (Gonzalez et al 2007;Mathieu-Rivet et al 2010).…”