Elsevier

Scientia Horticulturae

Volume 261, 5 February 2020, 108946
Scientia Horticulturae

Diurnal expression of Arabidopsis Gene homologs during daylength-regulated bulb formation in onion (Allium cepa L.)

https://doi.org/10.1016/j.scienta.2019.108946Get rights and content

Highlights

  • The paper provides new insights into the molecular basis for diurnal regulation of bulbing in onion.

  • Homologs of Arabidopsis photoperiodic flowering genes show similar diurnal expression in onion.

  • AcFt1, a bulb promoter and AcFt4, an inhibitor are expressed at different times of the day

  • Two new members of the onion CONSTANS are identified, including a candidate daylength regulator.

Abstract

Bulb initiation in long-day onion is regulated at the physiological level in a similar way to the photoperiodic regulation of flowering in Arabidopsis. This study establishes in onion, the diurnal time-course expression, in onion, of key genes particularly linked to circadian regulation in Arabidopsis. The long-day onion variety ‘Renate’ and the short-day (SD) onion variety ‘Hojem’ were used for these experiments. Onion plants were grown under natural LD conditions in the Phytobiology Glasshouse and immediately after bulbing they were transferred to two SANYO 2279 controlled environment cabinets for 10 d providing constant LD (16 h photoperiod including 8 h fluorescent followed by 8 h incandescent light) and constant short days (8 h photoperiod with fluorescent light). Five FLOWERING LOCUS T (FT) and three CONSTANS-LIKE (COL) genes were identified in onion, including two novel COL sequences through RNA-Seq analysis. The new AcCOL2 shows a diurnal pattern of expression similar to Arabidopsis CONSTANS (CO). Allium cepa FLAVIN-BINDING, KELCH REPEAT, F-BOX PROTEIN 1 (AcFKF1), Allium cepa GIGANTEA (AcGI) and AcCOL2 showed good diurnal expression patterns consistent with photoperiod sensing and regulation of AcFT1. All FT genes exhibited different diurnal expression patterns peaking at different times of the day. Notably, AcFT1 was expressed in the later part of the day which is very similar to the expression of Arabidopsis FT, while AcFT4 was expressed late in the night and the early morning in both Renate and Hojem varieties of onion, with the caveat that, AcFT4 is under less stringent daylength control in Hojem than in Renate. The timing of the peaks and expression pattern in both Renate F1 and Hojem suggest that AcFT5 may be under circadian or diurnal regulation under LD conditions and AcFT6 might not be circadian or diurnally regulated. These findings will help to understand the basis of the difference between responses of onions adapted to different latitudes, which is important for developing new varieties.

Introduction

Onion (Allium cepa L.) belongs to the family Alliaceae, is one of the most important vegetable and spice crops cultivated (Brewster, 1994; McCallum et al., 2001). Numerous onion cultivars have been developed for size, form, colour, pungency, storability, resistance to pests and pathogens, and climatic conditions (Griffiths et al., 2002). Onion is a monocotyledonous bulbous perennial (often biennial), outcrossing and highly heterozygous crop plant, which is propagated by seeds, bulbs or sets (Eady, 1995). An onion plant is composed of photosynthetic leaf blades, which arise alternately from a base plate, or small-flattened scales (bulb), which is the vegetative overwintering stage in the life cycle of the plant (Lancaster et al., 1996). Bulb formation in onions from different global regions is adapted to local environmental conditions, particularly the daylength (Cardoso and da Costa, 2003). Onions are classified as long-day, intermediate-day or short-day, depending on the minimum daily duration of light required for bulbing, also known as the critical daylength (Albert, 2016). Temperate onions require long days (LD) for bulbing whereas tropical onions will form bulbs in short days (SD) (Rashid et al., 2016). The life cycle of onion can be divided into three main stages, namely seedling growth and bulb formation in the first year and, following overwintering, flowering in the second year (Brewster et al., 1990). Bulb initiation will not occur during early seedling growth, sometimes referred to as the Juvenile phase, regardless of plants being exposed to favourable environmental conditions (Massiah, 2007). When the onion plant becomes mature and the daylength has reached a critical length, bulb formation is initiated (Lee et al., 2013). At this stage, onion leaves must be exposed continuously to an inductive photoperiod in order to initiate and complete bulbing (Brewster, 2008). The long-day onion cv. Renate requires at least 14 h of light to initiate bulbing (Rashid et al., 2016), whereas Hojem, a short-day cultivar requires at least 10 h to enable bulbing.

Arabidopsis flowering and onion bulb formation are both photoperiodically driven processes (Thomas et al., 2006), induced by LD, signal perception is in the leaf and response is at the apex. Sepals, petals, stamens and anthers are produced as the end product in Arabidopsis, whereas, a storage scale leaves are produced as the end product in onion (Summerfield et al., 1991). Arabidopsis flowering and onion bulb formation can be compared in terms of the involvement of phytochrome, and both processes are promoted by far-red light, through PHYA (Brewster et al., 1977). Flowering in Arabidopsis has been characterised at the molecular and genetic level and is regulated by 6 major separate pathways viz., photoperiodic, convergent autonomous, sucrose, gibberellin, temperature and light quality pathway (Jack, 2004; Thomas et al., 2006). For onion, the main environmental stimuli are photoperiod and temperature (Brewster et al., 1990), but these are mainly based on physiological rather than genetics analyses (Khokhar, 2017).

In this study we focus on the photoperiodic pathway, which is mediated by the circadian clock, an autonomous mechanism that generates endogenous rhythms in a 24 -h period in the leaf (Jackson, 2009) and is controlled by various feedback loops (Hayama and Coupland, 2003). Light plays an important role in the photoperiodic response in Arabidopsis and interacts with the circadian clock as part of the photoperiodic flowering pathway (Michael et al., 2003). In the leaf, light is perceived by different photoreceptors, both cryptochromes in blue light and phytochromes in red/ far-red light and inputs into the circadian clock (Devlin and Kay, 2000; Lin, 2002). Numerous key genes are involved in circadian regulation, where the clock derives the rhythmic expression of key genes FLAVIN-BINDING, KELCH REPEAT, F-BOX (FKF1), GIGANTEA (GI) and CONSTANS (CO). FKF1 and GI promote CO expression (Sawa et al., 2007) and this CO positively regulates FLOWERING LOCUS T (FT) (Jung et al., 2007). The FT protein is then translocated to the apical meristem through the phloem and forms a FT/FD (FLOWERING LOCUS D) complex (Abe et al., 2005; Pnueli et al., 2001; Purwestri et al., 2009; Taoka et al., 2011; Wigge et al., 2005). This complex activates the APETALA 1 (AP1) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) genes, which trigger LEAFY (LFY) gene expression and cause flowering at the floral apical meristem in Arabidopsis (Greg et al., 2015; Nakamichi, 2011; Yoo et al., 2005). In a previous study the expression of onion GI, FKF1 and ZTL homologs under SD and LD conditions was examined using quantitative reverse transcription-PCR (qRT-PCR), where the results showed that key genes namely GI, CO and FT controlling photoperiodic flowering in Arabidopsis are conserved in onion, and a role for these genes in the photoperiodic control of bulb initiation is predicted (Taylor et al., 2010). Also, Lee at al. (2013) identified 6 members of the FT family (FT1-6) in onion. They proposed that two of them, FT1 and FT4 acted to regulate bulbing, being promoter and inhibitor respectively, although they did not look at their circadian expression. This raised the question of how these genes are linked to the daylength-sensing system to establish the critical daylength in long-day and short-day onions. To address this question, experiments were designed to quantify the diurnal expression of FT, CO and other key genes in two onion cultivars with contrasting daylength responses, namely a long-day type cv. Renate and a short-day type cv. Hojem.

Section snippets

Materials and methods

This work has been conducted at the School of Life Sciences, the University of Warwick, Coventry, CV4 7AL, UK during the period from July 2013 to September 2016 to investigate the diurnal expression of Arabidopsis gene homologs during daylength-regulated bulb formation in onion (Allium cepa L.). The plant physiological experiments including growing of onion plants have been performed at the Phytobiology Facility and all laboratory analyses have been done at the School of Life Sciences Plant Lab

Transcriptome analysis and sequence comparison in Renate

An objective of the study was to identify and isolate a range of key genes hypothesised to be involved in bulbing in response to daylength. A combination of approaches was used, including identifying genes from EST databases, sequences from published work and through a transcriptome assembly. For the latter, RNA-seq analysis provided 12,604 differential expressed transcripts in LD leaf vs bulb, 13,665 in SD leaf vs bulb, 484 in SD leaf vs LD leaf and 964 in SD bulb vs LD bulb of onion.

Discussion

In this work we studied the expression patterns of putative onion homologs of Arabidopsis genes involved in the photoperiod regulation of flowering. Homology is the existence of shared common ancestry between a pair of structures, or genes, in different taxa (Pearson, 2013) and common rule of thumb is that two sequences are homologous if they are more than 30% identical over their entire lengths. Sequences that share more than 40% identity are very likely to be considered as high homology or

Declaration of competing interest

Dr. Md. Harun Ar Rashid and Professor Brian Thomas designed the experiments and wrote the paper. Dr. Md. Harun Ar Rashid conducted the experiments. The authors declare that there is no conflict of interests regarding the publication of this paper.

Declaration

The authors declare that the manuscript report is unpublished work and it is not under active consideration for publication elsewhere, nor been accepted for publication, nor been published in full or in part.

Acknowledgements

The authors are pleased to thank the Chancellors’ International Scholarship/Midland Integrative Biosciences Training Partnership (MIBTP) award at the University of Warwick, Coventry, CV4 7AL, UK for funding this research.

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    1

    Current affiliation: Department of Horticulture, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh.

    2

    The nucleotide sequences reported in this paper have been submitted to NCBI database (NCBI, 2016) with accession numbers (KY012331, KY012332, KY072874, KY072880, KY072881, KY072882).

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