Discussion
Accurate cell cycle progression is important for hypocotyl elongation and thickening, which requires precisely mediated APC/C activities. Several positive regulators of APC/C, SlCDC20 and SlCCS52genes were previously identified based on their homologous sequences to Arabidopsis, Medicago and human homologs (Mathieu-Rivet, Gevaudant, Sicard, et al., 2010). In this study, we isolated the negative regulatorSlUVI4 and new positive regulators, SlCDC20.3 ,SlCDC20.4 and SlCCS52A2 using published tomato genome sequence in solgenomics website (Tomato Genome, 2012). BothSlUVI4 and SlCCS52B had specifically high expressions in stems. Further analyses revealed that their transcript abundances correlated with cell cycle progression and hypocotyl elongation under different light conditions, and in MS salts and sugar treatments. They responded differently to exogenous hormones and environmental stresses to modulate cell cycle progression and hypocotyl elongation. Their overexpression lines in Arabidopsis and sluvi4 mutants had altered cell cycle progression and hypocotyl elongation. Therefore, we draw the conclusion that SlUVI4 and SlCCS52B coordinated cell cycle progression play important roles in the regulation of hypocotyl elongation.
UVI4 proteins are unique and conserved in the plant kingdom (d’Erfurth et al., 2009). Like most diploid Solanaceae species, tomato has only oneUVI4 gene in the genome indicating its central role in plant growth and development. In Brassicaceae species, a genome duplication brings about two homologous genes, UVI4 and OSD1 , which could be clearly distinguished via sequence similarity analysis (Mieulet et al., 2016). UVI4 and OSD1 mainly regulate mitosis and meiosis respectively, and knockout of both genes leads to the female gametophyte lethality (Bao & Hua, 2014; Hase, Trung, Matsunaga, & Tanaka, 2006; Heyman et al., 2011; Iwata et al., 2011). Here we presented results on tomato homologue SlUVI4 in the regulation of cell cycle progression during hypocotyl elongation, while its roles in the gametophyte development require further studies. CCS52 genes are activators of APC/C to promote the onset and progression of endoreduplication in plant organs (Cebolla et al., 1999; Vanstraelen et al., 2009). In tomato, only SlCCS52A1 (previously asCCS52A ) was reported to play important roles in fruit development through the regulation of endoreduplication (Mathieu-Rivet, Gevaudant, Sicard, et al., 2010). In Arabidopsis, UVI4 negatively regulates APC/C activity through the interaction with CDC20 and CCS52 proteins. SlUVI4 can interact with SlCCS52A1 and SlCCS52A2, but not with SlCCS52B. Whether or not SlUVI4 can interact with the conserved domain of SlCCS52B remains to be further explored. And here we want to emphasize that the similar spatial and temporal expression patterns of SlUVI4 andSlCCS52B imply important roles of their coordination in the regulation of tomato organ growth and development.
Hypocotyl elongation is based on cell division and expansion, which are maintained by APC/C activities. Based on our observations, the highest expression of SlCCS52 genes right after the seed gemination results in the most robust APC/C activities to promote hypocotyl elongation, which consequently ensures the successful emergence from soil. Down-regulation of SlCCS52A1 transcription led to the reduced plant heights and fruit size with decreased endoreduplication (Mathieu-Rivet, Gevaudant, Sicard, et al., 2010). Overexpression ofSlCCS52A1 in tomato resulted in the promotion of endoreduplication and abnormal growth phenotypes including impaired root and leaf growth (Mathieu-Rivet, Gevaudant, Cheniclet, Hernould, & Chevalier, 2010; Mathieu-Rivet, Gevaudant, Sicard, et al., 2010). Interestingly, overexpression lines of SlCCS52B in Arabidopsis generated in this study also had shorter hypocotyls with enhanced endoreduplication (Figure 11). Therefore, excessive APC/C activities triggered by overexpression of SlCCS52 genes requiresSlUVI4 -mediated inhibition machinery to fine-tune the APC/C activities and facilitate plant growth and development. In this case, tomato hypocotyl might grow faster when both SlUVI4 andSlCCS52B have higher transcript abundances, which proved to be true when tomato seedlings grew under different lights and in MS salts and sugar treatments. Light promotes cell division by activating photoreceptors to suppress cell division inhibitors (Okello, de Visser, Heuvelink, Marcelis, & Struik, 2016). Light inhibits the transcription of KRP1 , a CDK inhibitor, and activates cell cycle genes such asCDKB1;1 , CDKB1;2 and CYCA2;2 (Lopez-Juez et al., 2008). Under light conditions, the transcription of SlUVI4 andSlCCS52B is growth-dependent, and higher in younger hypocotyls. Their reduced transcription during the hypocotyl growth correlated with enhanced cell division suggesting their coordination in endoreduplication.
Exogenous application of ethylene precursor ACC stimulates cambial cell division in Populus (Love et al., 2009), which may be the reason of increased tomato hypocotyl thickness after ACC treatment (Figure 9C). Application of ACC promotes SlUVI4 transcription, whereas represses SlCCS52B transcription, which collectively results in the active cell division. However, we observed promotion of endoreduplication in hypocotyl cells, which is due to ethylene-induced DNA synthesis without cytokinesis through unknown mechanisms (Dan, Imaseki, Wasteneys, & Kazama, 2003). We observed the similar phenomena of hypocotyl elongation, cell cycle progression and transcriptions ofSlUVI4 and SlCCS52B under salt stress condition, suggesting that ethylene signaling is involved in salt responses. Ethylene drastically promotes hypocotyl elongation in nutrient-starved Arabidopsis seedlings under light condition, and might be a mediator of auxin-induced hypocotyl elongation (Smalle et al., 1997). High temperature promotes Arabidopsis hypocotyl elongation due to enhanced auxin synthesis or catabolism (Gray et al., 1998), which results in enhanced tomato hypocotyl elongation as well. High temperature induced promotion of endoreduplication in hypocotyl cells might be attributed to enhanced ethylene biosynthesis mediated by auxin (Kang, Newcomb, & Burg, 1971), which correlates with the repressed and promoted transcription of SlUVI4 and SlCCS52A2 in our study, respectively. However, the regulation machinery of SlUVI4 andSlCCS52A2 transcription by high temperature remains to be further studied.
The deletion mutant of SlUVI4 and overexpression lines ofSlCCS52B both have promoted endoreduplication in hypocotyl cells, indicating their antagonistic roles in cell cycle progression as the function of their homologues previously reported in Arabidopsis (Bao & Hua, 2014; Heyman et al., 2011; Iwata et al., 2011). SlCCS52Bfunction remains to be further studied using tomato mutants. Hypocotyl elongation is dependent on the active cell division and consequent cell expansion, which are regulated by SlUVI4 and SlCCS52B , respectively. Based on our study, we proposed a possible model ofSlUVI4 and SlCCS52B coordinated cell cycle progression (Figure S11). Under normal growth condition, light suppressesSlUVI4 and SlCCS52B coordinated cell cycle progression to inhibit hypocotyl elongation. MS salts and sugar promote SlUVI4and SlCCS52B coordinated cell cycle progression to improve hypocotyl elongation. Under stress condition, salt stress triggers ethylene-mediated repression of SlUVI4 and SlCCS52B to inhibit hypocotyl elongation. Heat stimulates auxin and its mediated ethylene production to modulate SlUVI4 and SlCCS52Bfunction to promote hypocotyl elongation.