Characterisation and Expression Analysis of LdSERK1, a Somatic Embryogenesis Gene in Lilium davidii var. unicolor.

The Lanzhou lily (Lilium davidii var. unicolor) is a variant of the Sichuan lily of the lily family and is a unique Chinese 'medicinal and food' sweet lily. Somatic cell embryogenesis of Lilium has played an important role in providing technical support for germplasm conservation, bulb propagation and improvement of genetic traits. Somatic embryogenesis receptor-like kinases (SERKs) are widely distributed in plants and have been shown to play multiple roles in plant life, including growth and development, somatic embryogenesis and hormone induction. Integrating the results of KEGG enrichment, GO annotation and gene expression analysis, a lily LdSERK1 gene was cloned. The full-length open reading frame of LdSERK1 was 1875 bp, encoding 624 amino acids. The results of the phylogenetic tree analysis showed that LdSERK1 was highly similar to rice, maize and other plant SERKs. The results of the subcellular localisation in the onion epidermis suggested that the LdSERK1 protein was localised at the cell membrane. Secondly, we established the virus-induced gene-silencing (VIGS) system in lily scales, and the results of LdSERK1 silencing by Tobacco rattle virus (TRV) showed that, with the down-regulation of LdSERK1 expression, the occurrence of somatic embryogenesis and callus tissue induction in scales was significantly reduced. Finally, molecular assays from overexpression of the LdSERK1 gene in Arabidopsis showed that LdSERK1 expression was significantly enhanced in the three transgenic lines compared to the wild type, and that the probability of inducing callus tissue in seed was significantly higher than that of the wild type at a concentration of 2 mg/L 2,4-D, which was manifested by an increase in the granularity of the callus tissue.

Epigenetic silencing of callose synthase by VIL1 promotes bud-growth transition in lily bulbs.

In plants, restoring intercellular communication is required for cell activity in buds during the growth transition from slow to fast growth after dormancy release. However, the epigenetic regulation of this phenomenon is far from understood. Here we demonstrate that lily VERNALIZATION INSENSITIVE 3-LIKE 1 (LoVIL1) confers growth transition by mediating plasmodesmata opening via epigenetic repression of CALLOSE SYNTHASE 3 (LoCALS3). Moreover, we found that a novel transcription factor, NUCLEAR FACTOR Y, SUBUNIT A7 (LoNFYA7), is capable of recruiting the LoVIL1-Polycomb Repressive Complex 2 (PRC2) and enhancing H3K27me3 at the LoCALS3 locus by recognizing the CCAAT cis-element (Cce) of its promoter. The LoNFYA7-LoVIL1 module serves as a key player in orchestrating the phase transition from slow to fast growth in lily bulbs. These studies also indicate that LoVIL1 is a suitable marker for the bud-growth-transition trait following dormancy release in lily cultivars.

LhANS-rr1, LhDFR, and LhMYB114 Regulate Anthocyanin Biosynthesis in Flower Buds of Lilium 'Siberia'.

The bulb formation of Lilium is affected by many physiological and biochemical phenomena, including flower bud differentiation, starch and sucrose accumulation, photoperiod, carbon fixation, plant hormone transduction, etc. The transcriptome analysis of flower buds of Lilium hybrid 'Siberia' at different maturity stages showed that floral bud formation is associated with the accumulation of anthocyanins. The results of HPLC-MS showed that cyanidin is the major anthocyanin found in Lilium 'Siberia'. Transcriptome KEGG enrichment analysis and qRT-PCR validation showed that two genes related to flavonoid biosynthesis (LhANS-rr1 and LhDFR) were significantly up-regulated. The functional analysis of differential genes revealed that LhMYB114 was directly related to anthocyanin accumulation among 19 MYB transcription factors. Furthermore, the qRT-PCR results suggested that their expression patterns were very similar at different developmental stages of the lily bulbs. Virus-induced gene silencing (VIGS) revealed that down-regulation of LhANS-rr1, LhDFR, and LhMYB114 could directly lead to a decrease in anthocyanin accumulation, turning the purple phenotype into a white color. Moreover, this is the first report to reveal that LhMYB114 can regulate anthocyanin accumulation at the mature stage of lily bulbs. The accumulation of anthocyanins is an important sign of lily maturity. Therefore, these findings have laid a solid theoretical foundation for further discussion on lily bulb development in the future.

Transcriptome Analysis Reveals the Molecular Regularity Mechanism Underlying Stem Bulblet Formation in Oriental Lily 'Siberia'; Functional Characterization of the LoLOB18 Gene.

The formation of underground stem bulblets in lilies is a complex biological process which is key in their micropropagation. Generally, it involves a stem-to-bulblet transition; however, the underlying mechanism remains elusive. It is important to understand the regulatory mechanism of bulblet formation for the reproductive efficiency of Lilium. In this study, we investigated the regulatory mechanism of underground stem bulblet formation under different conditions regarding the gravity point angle of the stem, i.e., vertical (control), horizontal, and slanting. The horizontal and slanting group displayed better formation of bulblets in terms of quality and quantity compared with the control group. A transcriptome analysis revealed that sucrose and starch were key energy sources for bulblet formation, auxin and cytokinin likely promoted bulblet formation, and gibberellin inhibited bulblet formation. Based on transcriptome analysis, we identified the LoLOB18 gene, a homolog to AtLOB18, which has been proven to be related to embryogenic development. We established the stem bud growth tissue culture system of Lilium and silenced the LoLOb18 gene using the VIGS system. The results showed that the bulblet induction was reduced with down-regulation of LoLOb18, indicating the involvement of LoLOb18 in stem bulblet formation in lilies. Our research lays a solid foundation for further molecular studies on stem bulblet formation of lilies.