Abstract
Bulb onion is an important vegetable crop cultivated worldwide for salad and culinary purposes. Onions have a biennial life cycle; during the first year the bulb is formed followed by flowering in the second year. The bulb is a modified underground stem formed by a thickening of leaf sheaths in response to day length, whereas flowering occurs once vernalization requirement has been fulfilled. The physiological basis of day length sensing and vernalization is well studied in bulb onion but little information is available at molecular level. Therefore, this project was planned to investigate the molecular mechanism of bulbing and flowering regulation in bulb onion.
Development and utilization of genomic resources in bulb onion are hampered due to its highly heterozygous nature and large genome. To overcome these challenges, a transcriptome dataset was developed using vegetative and reproductive tissues from the homozygous double haploid line ‘CUDH-2107’ (chapter 2). A gene ontology analysis was carried out to assign a putative role to transcripts, and differential expression analysis was conducted to understand the expression pattern of transcripts over different developmental stages. Further, to demonstrate the potential of the ‘CUDH-2107’ transcriptome catalogue for understanding the genetic and molecular basis of various traits, orthologues of rice genes involved in male fertility and flower development were identified in ‘CUDH-2107’ transcriptome dataset. It was found that MADS-box genes involved in flower development were expressed in different reproductive tissues indicating that the ABCDE model of flower development might be conserved in bulb onion.
The genes involved in day length sensing and flowering have been studied in monocot grasses, but little information is available for bulb onion and other Allium species. In chapter 3, transcriptome datasets of bulb onion and other Allium species were explored using comparative genomics approaches. It was found that various photoreceptors and circadian clock genes were conserved between different Allium species and grasses. Different photoreceptors and circadian clock genes follows a diurnal expression patterns and respond to change in day length. Further, bulb onion FT genes involved in bulbing (AcFT1 and AcFT4) and flowering (AcFT2) regulation show a high degree of sequence conservation and a close relationship with other Allium species.
The two FT genes are involved in bulbing regulation act antagonistically to control bulbing in response to day length. Bulb onion cultivars adapted at different latitudes exhibit a wide variation for critical day length required to form bulbs. However, it is not known how day length sensitivity differences between short day (SD) and long day (LD) onion varieties occurs. In chapter 4 it was shown that in addition to day length, optimum light quality (far-red) was also involved in AcFT1 and AcFT4 regulation to control bulbing. Further, results imply that the same FTs (AcFT1 and AcFT4) regulate bulbing in SD and LD onions but these respond differently to different critical day lengths. Upstream photoperiodic pathway genes (photoreceptors and circadian clock) exhibit differential expression patterns between SD and LD onions, suggesting that a clock gene, such as AcFKF1, might be responsible for differences in day length sensitivity of bulb onion cultivars adapted to different latitudes.
In Arabidopsis, the transition from the vegetative to reproductive phase is regulated by FT, which forms a florigen activation complex (FAC) at the shoot apical meristem (SAM) and activates floral identity genes, such as APETALA1 (AP1) to impart floral identity. However, the role of AP1 genes is not known in storage organ development. In chapter 5, two AP1 genes were identified in bulb onion (AcVRN1 and AcAP1) and their role in the photoperiodic induction of bulbing was studied. AcVRN1 was not associated with bulbing but AcAP1 was only expressed in bulbing plants that express AcFT1. Furthermore, AcAP1 was not expressed in transgenic bulb onions over-expressing AcFT4, a repressor of AcFT1, but was expressed in control bulbing plants. However, it is not yet known whether AcFT4 represses AcAP1 directly or indirectly by repressing AcFT1 which would might activate AcAP1. As AcAP1 was upregulated in the basal plate (shoot apical meristem) during bulb initiation, we hypothesise that AcFT1 is produced in the leaves and moves to the basal plate to activate AcAP1 to initiate bulb formation. The sequence conservation and protein modelling analysis suggest that AcFT1 can interact with Ac14-3-3 and might form a FAC-like complex to regulate bulbing.
The molecular mechanism of vernalization sensing and flowering is well understood in temperate grasses. In bulb onion, it has been discovered that the FT gene (AcFT2) respond to vernalization, but information is lacking about its regulation by other genes and AcFT2 role in flowering induction. In chapter 6, candidate vernalization pathway genes, REPRESSOR OF VERNALIZATION1 (RVR1) and VERNALIZATION1 (VRN1) have been identified and characterized. AcRVR1 was expressed in non-vernalized onions and downregulated due to cold, whereas AcVRN1 and AcFT2 were expressed at low levels in non-vernalized onions and at high levels following cold treatment. AcVRN1 and AcFT2 exhibit a quantitative response to the duration of cold exposure and were also expressed in different floral stages. Sequence conservation and protein modelling analysis suggest that AcFT2 might bind with Ac14-3-3 proteins and form FAC to specify floral identity.
In summary, the results of this thesis indicate that duplication of the FT and AP1 genes have resulted in two pathways; one which triggers bulb formation and involves the day length activation of AcFT1 (and repression of AcFT4) and AcAP1; and another pathway where the vernalization results in the activation of AcFT2 and the related AP1 gene, AcVRN1 to induce flowering.