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.