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  • Eukaryotic cell cycle machinery is


    Eukaryotic cell cycle machinery is thought to appropriately modulate cell proliferation and cell expansion in various developmental stages and environmental conditions. The core feature of cell-cycle control is the fluctuation of CDK activity, which is regulated by three interactive and conserved mechanisms: (1) the synthesis and destruction of cyclin proteins; (2) the assembly, inhibitory and activating phosphorylation of CDK-cyclin complex; and (3) binding inhibitory proteins (Alberts et al., 2008; Inagaki and Umeda, 2011; Lents and Baldassare, 2016). Hence, the sequences and motifs of plant CDKs are quite conserved, and phosphorylation sites, substrate-binding site, CDK/cyclin interface site and activation loop domain were also identified in the VcCDK deduced amino beta lapachone sequences (Fig. 4). Comparatively, Vccyclins were more specific (Table 2 and Fig. 4), indicated cyclins could play distinct roles in cell cycle progression, fruit growth and development, as well as responding to environmental change (Bulankova et al., 2013; Menges et al., 2005; Vandepoele et al., 2002; Zhang et al., 2014). Although VcCDKs were conserved, and Vccyclins were flexible from other animals and plants, its biological functions were predicted reserved. For instance, CDKBs are plant-specific CDKs, which interact with CycA and CycB, positively regulate G2-to-M transition, and cell production and fruit growth (Apri et al., 2014; Malladi and Hirst, 2010; Scofield et al., 2014). In our study, two noticeable peaks were displayed in the VcCDKB2;2 expression pattern, consistent with the higher expression of VcCycA2L and VcCycB2L in the early development of flower buds, and the expression of VcCycD3;1L, VcCycT1;4L and VcCDKE1 at the first rapid fruit growth stage, separately (Fig. 5). Hence, VcCDKB2;2, VcCycA2L, VcCycB2L, VcCycD3;1L, VcCycT1;4L and VcCDKE1 might be the positive regulators for cell production in the flower bud enlargement and early fruit development. In addition, the expression levels of these genes were decreased dramatically, even undetectable from stage S4 till mature, indicated these genes not only promoted cell division, but facilitated divided cells entering into endoreduplication cycle. However, further studies are need to carry out by utilizing flow cytometry and/or cellular imaging (De Veylder et al., 2011; Pirrello et al., 2014). Plant hormones are thought as key regulators for fruit set and growth (Inagaki and Umeda, 2011; Pascual et al., 2009; Scofield et al., 2014; Vergara et al., 2017; Zhang et al., 2010; Zifkin et al., 2012). Trans- and cis-zeatin-O-glucosides, free indole-3-acetic acid (IAA) and abscisic acid (ABA) were major phytohormones during V. corymbosum \'Rubel\' fruit development, which were mainly accumulated at the stage I, II and III of double-sigmoid pattern, respectively (Zifkin et al., 2012). Correspondingly, the expression tendency of VcCycD3;1L, VcCycT1;4L, VcCDKB2;2 and VcCDKE1 in this study was in accordance with cytokine accumulation, and was down-regulated by ABA, while VcCDKF4L expression was followed the change of auxin and abscisic acid contents, indicated that these genes could respond to phytohormones (Flaishman et al., 2015; Vergara et al., 2017). Among these phytohormone-responsive genes, plant D-type cyclins were regarded as positive regulators for G0-to-G1 and G1-to-S transitions in response to developmental and environmental signals, and CycD3 is a key target of cytokinin in its regulation of cell cycle (Joubes et al., 2000; Zhang et al., 2010). Combined with the gene expression patterns during flower bud and fruit growth, VcCycB2L, VcCycD3;1L, VcCycP3;1L, VcCDKB2;2, VcCDKD3L, VcCDKE1 and VcCDKF4L might be involved in perceiving the growth and environmental signals, and promoting fruit cell production, endocycle and cell expansion. However, VcCycH1;1, VcCDKC1L and VcCDKG2L expression levels were relatively low, and exhibited irregular alteration, indicated these genes might not act on V. corymbosum flower bud and fruit development directly, but affected the growth circuitously.