Original research
The RING Finger Protein NtRCP1 Is Involved in the Floral Transition in Tobacco (Nicotiana tabacum)

https://doi.org/10.1016/j.jgg.2015.03.010Get rights and content

Abstract

The transition from the vegetative phase to the reproductive phase is a major developmental process in flowering plants. The underlying mechanism controlling this cellular process remains a research focus in the field of plant molecular biology. In the present work, we identified a gene encoding the C3H2C3-type RING finger protein NtRCP1 from tobacco BY-2 cells. Enzymatic analysis demonstrated that NtRCP1 is a functional E3 ubiquitin ligase. In tobacco plants, expression level of NtRCP1 was higher in the reproductive shoot apices than in the vegetative ones. NtRCP1-overexpressing plants underwent a more rapid transition from the vegetative to the reproductive phase and flowered markedly earlier than the wild-type control. Histological analysis revealed that the shoot apical meristem of NtRCP1-overexpressing plants initiated inflorescence primordia precociously compared to the wild-type plant due to accelerated cell division. Overexpression of NtRCP1 in BY-2 suspension cells promoted cell division, which was a consequence of the shortened G2 phase in the cell cycle. Together, our data suggest that NtRCP1 may act as a regulator of the phase transition, possibly through its role in cell cycle regulation, during vegetative/reproductive development in tobacco plant.

Introduction

The transition from vegetative to reproductive growth (the floral transition) is a critical developmental process in flowering plants. During the past two decades, great progress has been made in revealing the molecular mechanisms that regulate the floral transition in higher plants. In Arabidopsis, five major pathways involved in the floral transition have been identified, namely, the photoperiod, vernalization, gibberellin, autonomous, and age pathways (Amasino and Michaels, 2010, Wang, 2014). Various components in these pathways have been well characterized as important regulators of flowering time, including CONSTANS (CO), FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), LEAFY (LFY), and FLOWERING LOCUS C (FLC).

The really interesting new gene (RING) family is the largest group of E3 ligases in the ubiquitin-mediated protein degradation pathway. These enzymes participate in multiple plant biological functions, including development, hormone responses, abiotic and biotic stress responses, and cell cycle regulation (Xie et al., 2002, Zhang et al., 2007, Johnson et al., 2012, Luo et al., 2012, Peng et al., 2013, Yu et al., 2013). Several studies demonstrate that RING finger proteins play an important role in the control of photoperiodic flowering (Piñeiro and Jarillo, 2013). In Arabidopsis, SINAT5 interacts directly with LHY and inhibits flowering, while COP1, HOS1, RFI2 and DNF negatively regulate flowering time by control the expression level of CO gene (Chen and Ni, 2006, Liu et al., 2008, Morris et al., 2010, Park et al., 2010, Lazaro et al., 2012). In rice, the COP1 ortholog PPS could regulate vegetative-reproductive phase change by inhibition expression level of RAP1B, a floral meristem identity protein, resulting in a flowering delay (Tanaka et al., 2011).

The shoot apical meristem (SAM) is a group of stem cells at the tip of the shoot axis, which generate leaves during the vegetative growth stage and flowers in the reproductive growth stage (Poethig, 2003, Wang, 2014). During the floral transition stage, an early event observed in many flowering plants is the acceleration of cell division at the SAM (Lyndon and Francis, 1984, Lyndon and Battey, 1985, Bernier, 1988, Francis, 1992, Francis and Herbert, 1993, Jacqmard et al., 2003). Base on this common feature, it has been proposed that an increase in the cell division rate is a prerequisite for the morphological changes that occur at the SAM during the floral transition (Francis, 1992, Bernier, 1997, Jacqmard et al., 2003, Kwiatkowska, 2008). In line with this morphological evidence, flower bud formation occurs earlier in tobacco ectopically expressing Arabidopsis CYCD2 and CYCD3 than the wild type, and the structural organization of the SAM is dramatically altered in these transgenic plants (Boucheron et al., 2005). Although the association of cell cycle regulation with floral induction is well recognized, the molecular basis for the collection between these two processes remains elusive.

In this study, we identified a C3H2C3-type RING finger protein from tobacco BY-2 cells and analyzed the physiological role of this protein in tobacco plants using a reverse genetics strategy. The results reveal that NtRCP1 is involved in the control of the vegetative-reproductive phase transition in tobacco plants, perhaps by regulating the cell division cycle at SAM.

Section snippets

Identification of NtRCP1 from tobacco BY-2 cells and protein structure analysis

In a previous study, we identified a number of cytokinesis-related cDNAs from tobacco BY-2 cells using fission yeast as a functional system (Yu et al., 2007). Ectopic overexpression of one of these cDNAs in yeast cells caused dramatic defects in cell plate formation. As shown in Fig. 1A, the transgenic yeast cells were highly elongated and each contained 2–8 nuclei, demonstrating that overexpression of this plant gene has a strong effect on cell division in yeast.

Sequence analysis indicated

Discussion

Growing evidence indicates that RING finger proteins participate in the regulation of flowering time in higher plants. In the long-day plant Arabidopsis, the RING finger protein AtRING1A regulates flowering through repressing MADS AFFECTING FLOWERING genes, and the E3 ligase HOS1 regulates flowering by mediating CO degradation (Jung et al., 2012, Lazaro et al., 2012, Shen et al., 2014). In this study, we characterized the function of the RING finger protein NtRCP1 in tobacco plants. The results

Yeast strain and fluorescence microscopy

The fission yeast Schizosaccharomyces pombe (h, leu1-32) was used in this study. S. pombe was maintained according to standard methods (Alfa et al., 1993). For microscopy, DNA and cell wall materials were visualized using 4′, 6-diamidino-2-phenylindole (DAPI, Roche Diagnostics Hong Kong Ltd., Hong Kong) and calcofluor (Sigma, USA) staining after fixation with 70% ethanol. Photographs were taken using a UV light microscope (Olympus BX51, Tokyo, Japan) with a 100× objective lens.

Plant material and cell culture

Tobacco (

Acknowledgments

We are grateful to Prof. Qi Xie and Dr. Qing-Zhen Zhao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for their kindly help on E3 ligase activity analysis. This work was supported by the Natural Science Foundation of China (Grant Nos. 31100870 and 30800556).

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