WUSCHEL-Related Homeobox Genes Cooperate with Cytokinin Signalling to Promote Bulbil Formation in Lilium lancifolium

The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium. Based on our previously obtained transcriptome data, we screened two WUSCHCEL-related homeobox (WOX) genes closely related to bulbil formation, LlWOX9 and LlWOX11. However, the biological functions and regulatory mechanisms of LlWOX9 and LlWOX11 are unclear. In this study, we cloned the full-length coding sequences of LlWOX9 and LlWOX11. Transgenic Arabidopsis showed increased branch numbers, and the overexpression of LlWOX9 and LlWOX11 in stem segments promoted bulbil formation, while the silencing of LlWOX9 and LlWOX11 inhibited bulbil formation, indicating that LlWOX9 and LlWOX11 are positive regulators of bulbil formation. Cytokinins acting through type-B response regulators (type-B RRs) could bind to the promoters of LlWOX9 and LlWOX11 and promote their transcription. LlWOX11 could enhance cytokinin pathway signalling by inhibiting the transcription of type-A LlRR9. Our study enriches the understanding of the regulation of plant development by the WOX gene family and lays a foundation for further research on the molecular mechanism of bulbil formation in lily.

Alteration of Flower Color in Viola cornuta cv. “Lutea Splendens” through Metabolic Engineering of Capsanthin/Capsorubin Synthesis

Flower color is an important characteristic that determines the commercial value of ornamental plants. The development of modern biotechnology methods such as genetic engineering enables the creation of new flower colors that cannot be achieved with classical methods of hybridization or mutational breeding. This is the first report on the successful Agrobacterium-mediated genetic transformation of Viola cornuta L. The hypocotyl explants of cv. “Lutea Splendens” variety with yellow flowers were transformed with A. tumefaciens carrying empty pWBVec10a vector (Llccs−) or pWBVec10a/CaMV 35S::Llccs::TNos vector (Llccs+) for capsanthin/capsorubin synthase gene (Llccs) from tiger lily (Lilium lancifolium). A comparative study of shoot multiplication, rooting ability during culture in vitro, as well as phenotypic characteristics of untransformed (control) and transgenic Llccs− and Llccs+ plants during ex vitro growth and flowering is presented. Successful integration of Llccs transgene allows the synthesis of red pigment capsanthin in petal cells that gives flowers different shades of an orange/reddish color. We demonstrate that the ectopic expression of Llccs gene in ornamental plants, such as V. cornuta “Lutea Splendens” could successfully be used to change flower color from yellow to different shades of orange.

Functional characterization of Lilium lancifolium cold-responsive Zinc Finger Homeodomain (ZFHD) gene in abscisic acid and osmotic stress tolerance

Background. We have previously performed an analysis of the cold-responsive transcriptome in the mature leaves of tiger lily (Lilium lancifolium) by gene co-expression network identification. The results has revealed that a ZFHD gene, notated as encoding zinc finger homeodomain protein, may play an essential regulating role in tiger lily response to cold stress. Methods. A further investigation of the ZFHD gene (termed as LlZFHD4) responding to osmotic stresses, including cold, salt, water stresses, and abscisic acid (ABA) was performed in this study. Based on the transcriptome sequences, the coding region and 5′ promoter region of LlZFHD4 were cloned from mature tiger lily leaves. Stress response analysis was performed under continuous 4 °C, NaCl, PEG, and ABA treatments. Functional characterization of LlZFHD4 was conducted in transgenic Arabidopsis, tobacco, and yeast. Results. LlZFHD4 encodes a nuclear-localized protein consisting of 180 amino acids. The N-terminal region of LlZFHD4 has transcriptional activation activity in yeast. The 4 °C, NaCl, PEG, and ABA treatments induced the expression of LlZFHD4. Several stress- or hormone-responsive cis-acting regulatory elements (T-Box, BoxI. and ARF) and binding sites of transcription factors (MYC, DRE and W-box) were found in the core promoter region (789 bp) of LlZFHD4. Also, the GUS gene driven by LlZFHD4 promoter was up-regulated by cold, NaCl, water stresses, and ABA in Arabidopsis. Overexpression of LlZFHD4 improved cold and drought tolerance in transgenic Arabidopsis; higher survival rate and better osmotic adjustment capacity were observed in LlZFHD4 transgenic plants compared to wild type (WT) plants under 4 °C and PEG conditions. However, LlZFHD4 transgenic plants were less tolerant to salinity and more hypersensitive to ABA compared to WT plants. The transcript levels of stress- and ABA-responsive genes were much more up-regulated in LlZFHD4 transgenic Arabidopsis than WT. These results indicate LlZFHD4 is involved in ABA signaling pathway and plays a crucial role in regulating the response of tiger lily to cold, salt and water stresses.

Effects of Continuous Cropping of Lilium Lancifolium on Rhizosphere Soil Physical and Chemical Properties and Fungal Community Structure

Lilium lancifolium is an important economic crop in Huoshan county of Anhui province, China. Continuous cropping obstacles serious affect the yield and quality of L. lancifolium. At present, the effect of the continuous cropping of L. lancifolium on soil fungal community structure is not clear. In this study, Illumina MiSeq was used to study the fungi of the rhizosphere soil associated with L. lancifolium subjected to three treatments: no continuous cropping, continuous cropping for 3 years, and continuous cropping for 5 years. The results showed that continuous cropping of L. lancifolium could increase the fungal richness and diversity in the rhizosphere to different degrees. Ascomycota was the dominant phylum, and its abundance increased after continuous cropping. In addition, the abundance of beneficial fungi, such as Chaetomium, decreased, and the abundance of harmful fungi, such as Fusarium and Colletotrichum, greatly increased with the duration of continuous cropping. Overall, continuous cropping changed the composition of soil fungal communities, reduced the abundance of beneficial fungi, and increased the abundance of harmful fungi. Thus, continuous cropping increased the potential for soil-borne diseases and endangered the bulb growth of L. lancifolium.

Full-Length Transcriptome Sequencing and EST-SSR marker development of tiger lily (Lilium lancifolium Thunb.)

It is well known that transcriptional diversity plays important roles in plant biological regulation. But for the difficulty in full-length transcripts obtainment, the available tiger lily (Lilium lancifolium Thunb) transcriptome characterization are still not complete. To improve the integrity of tiger lily transcriptome information, (SMRT PacBio single-molecule long-read sequencing technology) was employed to accomplish the whole transcriptome profiling. A total of 815,624 CCS (Circular Consensus Sequence) reads with mean length of 1,295 bp were obtained. Based on these transcripts, 61,744 reads were full-length reads containing both the 5’ primer, 3’ primer and the poly (A) tail and 3,319 EST-derived SSRs were developed from 2968 unigenes. With the obtained informative reference transcriptome,768 transcription factors and 6,852 long non-coding RNAs were identified, providing a comprehensive framework of the transcriptional regulation network. Of all the annotated transcripts, 15,608 were distributed into 25 various Clusters of euKaryotic Orthologous Groups (KOG), and 10,706 unigenes were categorized into 52 functional groups which were divided into three categories. These results would provide a comprehensive set of reference transcripts and further improve our understanding of the tiger lily transcriptomes.

Cytokinin Type-B Response Regulators Promote Bulbil Initiation in Lilium lancifolium

The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium whose development is promoted by cytokinins. Type-B response regulators (RRs) are critical regulators that mediate primary cytokinin responses and promote cytokinin-induced gene expression. However, the function of cytokinin type-B Arabidopsis RRs (ARRs) in regulating bulbil formation is unclear. In this study, we identified five type-B LlRRs, LlRR1, LlRR2, LlRR10, LlRR11 and LlRR12, in L. lancifolium for the first time. The five LlRRs encode proteins of 715, 675, 573, 582 and 647 amino acids. All of the regulators belong to the B-I subfamily, whose members typically contain a conserved CheY-homologous receiver (REC) domain and an Myb DNA-binding (MYB) domain at the N-terminus. As transcription factors, all five type-B LlRRs localize at the nucleus and are widely expressed in plant tissues, especially during axillary meristem (AM) formation. Functional analysis showed that type-B LlRRs are involved in bulbil formation in a functionally redundant manner and can activate LlRR9 expression. In summary, our study elucidates the process by which cytokinins regulate bulbil initiation in L. lancifolium through type-B LlRRs and lays a foundation for research on the molecular mechanism of bulbil formation in the lily.

The extraction of phenolic acids and polysaccharides from Lilium lancifolium Thunb. using a deep eutectic solvent

This study provided a new application of deep eutectic solvents (DESs) in the simultaneous extraction of phenolic acids and polysaccharides from the bulbs of Lilium lancifolium Thunb.