LiNAC100 contributes to linalool biosynthesis by directly regulating LiLiS in Lilium 'Siberia'.

MAIN CONCLUSION: The transcription factor LiNAC100 has a novel function of regulating floral fragrance by directly regulating linalool synthase gene LiLiS. Lilium 'Siberia', an Oriental hybrid, is renowned as both a cut flower and garden plant, prized for its color and fragrance. The fragrance comprises volatile organic compounds (VOCs), primarily monoterpenes found in the plant. While the primary terpene synthases in Lilium 'Siberia' were identified, the transcriptional regulation of these terpene synthase (TPS) genes remains unclear. Thus, understanding the regulatory mechanisms of monoterpene biosynthesis is crucial for breeding flower fragrance, thereby improving ornamental and commercial values. In this study, we isolated a nuclear-localized LiNAC100 transcription factor from Lilium 'Siberia'. The virus-induced gene silencing (VIGS) of LiNAC100 was found to down-regulate the expression of linalool synthase gene (LiLiS) and significantly inhibit linalool synthesis. Conversely, transient overexpression of LiNAC100 produced opposite effects. Additionally, yeast one-hybrid and dual-luciferase assays confirmed that LiNAC100 directly activates LiLiS expression. Our findings reveal that LiNAC100 plays a key role in monoterpene biosynthesis in Lilium 'Siberia', promoting linalool synthesis through the activation of LiLiS expression. These results offer insights into the molecular mechanisms of terpene biosynthesis in Lilium 'Siberia' and open avenues for biotechnological enhancement of floral scent.

Lily LlHSFC2 coordinates with HSFAs to balance heat stress response and improve thermotolerance.

Heat stress transcription factors (HSFs) are core regulators of plant heat stress response. Much research has focused on class A and B HSFs, leaving those of class C relatively understudied. Here, we reported a lily (Lilium longiflorum) heat-inducible HSFC2 homology involved in thermotolerance. LlHSFC2 was located in the nucleus and cytoplasm and exhibited a repression ability by binding heat stress element. Overexpression of LlHSFC2 in Arabidopsis, tobacco (Nicotiana benthamiana), and lily, all increased the thermotolerance. Conversely, silencing of LlHSFC2 in lily reduced its thermotolerance. LlHSFC2 could interact with itself, or interact with LlHSFA1, LlHSFA2, LlHSFA3A, and LlHSFA3B of lily, AtHSFA1e and AtHSFA2 of Arabidopsis, and NbHSFA2 of tobacco. LlHSFC2 interacted with HSFAs to accelerate their transactivation ability and act as a transcriptional coactivator. Notably, compared with the separate LlHSFA3A overexpression, co-overexpression of LlHSFC2/LlHSFA3A further enhanced thermotolerance of transgenic plants. In addition, after suffering HS, the homologous interaction of LlHSFC2 was repressed, but its heterologous interaction with the heat-inducible HSFAs was promoted, enabling it to exert its co-activation effect for thermotolerance establishment and maintenance. Taken together, we identified that LlHSFC2 plays an active role in the general balance and maintenance of heat stress response by cooperating with HSFAs, and provided an important candidate for the enhanced thermotolerance breeding of crops and horticulture plants.

Identification and Expression Analysis of Putative Sugar Transporter Gene Family during Bulb Formation in Lilies.

Sugar transporters play important roles in plant growth and development, flowering and fruiting, as well as responses to adverse abiotic and biotic environmental conditions. Lilies (Lilium spp.) are some of the most representative ornamental bulbous flowers. Sugar metabolism is critical for bulb formation in lilies; therefore, clarifying the amount and expression pattern of sugar transporters is essential for further analyzing their roles in bulb formation. In this study, based on the transcriptome data of the Lilium Oriental hybrid 'Sorbonne' and Lilium × formolongi, a total of 69 and 41 sugar transporters were identified in 'Sorbonne' and Lilium × formolongi, respectively, by performing bioinformatics analysis. Through phylogenetic analysis, monosaccharide transporters (MSTs) can be divided into seven subfamilies, sucrose transporters (SUTs) can be divided into three subgroups, and sugars will eventually be exported transporters (SWEETs) can be divided into four clades. According to an analysis of conserved motifs, 20, 14, and 12 conserved motifs were predicted in MSTs, SUTs, and SWEETs, respectively. A conserved domain analysis showed that MSTs and SUTs contained a single domain, whereas most of the SWEETs harbored two MtN3/saliva domains, also known as a PQ-loop repeat. The LohINT1, which was predicted to have a smaller number of transmembrane structural domains, was cloned and analyzed for subcellular localization. It was found that the LohINT1 protein is mainly localized in the cell membrane. In addition, the expression analysis indicated that 22 LohMSTs, 1 LohSUTs, and 5 LohSWEETs were upregulated in 'Sorbonne' 1 day after scale detachment treatment, suggesting that they may regulate the initiation of the bulblet. A total of 10 LflMSTs, 1 LflSUTs, and 6 LflSWEETs were upregulated 4~6 months after sowing, which corresponds to the juvenile-to-adult transition phase of Lilium × formolongi, suggesting that they may also play a role in the accompanying bulb swelling process. Combined with quantitative real-time PCR (qRT-PCR) analysis, LohSTP8 and LohSTP12 were significantly overexpressed during the extremely early stage of bulblet initiation, and LflERD6.3 was significantly overexpressed during the growth of the underground bulblet, suggesting that they may be key sugar transporters in the formation of lily bulbs, which needs further functional verification.

Transcriptome Analysis Reveals Coexpression Networks and Hub Genes Involved in Papillae Development in Lilium auratum.

Lilium is a genus of important ornamental plants with many colouring pattern variations. Lilium auratum is the parent of Oriental hybrid lilies. A typical feature of L. auratum is the presence of red-orange special raised spots named papillae on the interior tepals. Unlike the usual raised spots, the papillae are slightly rounded or connected into sheets and usually have hairy tips. To elucidate the potential genes regulating papillae development in L. auratum, we performed high-throughput sequencing of its tepals at different stages. Genes involved in the flavonoid biosynthesis pathway were significantly enriched during the colouration of the papillae, and CHS, F3H, F3'H, FLS, DFR, ANS, and UFGT were significantly upregulated. To identify the key genes involved in the papillae development of L. auratum, we performed weighted gene coexpression network analysis (WGCNA) and further analysed four modules. In total, 51, 24, 1, and 6 hub genes were identified in four WGCNA modules, MEbrown, MEyellow, MEpurple, and MEred, respectively. Then, the coexpression networks were constructed, and important genes involved in trichome development and coexpressed with anthocyanin biosynthesis genes, such as TT8, TTG1, and GEM, were identified. These results indicated that the papillae are essentially trichomes that accumulate anthocyanins. Finally, we randomly selected 12 hub genes for qRT-PCR analysis to verify the accuracy of our RNA-Seq analysis. Our results provide new insights into the papillae development in L. auratum flowers.

Functional Study on the Key Gene LaLBD37 Related to the Lily Bulblets Formation.

Oriental hybrid lilies, known for their vibrant colors, diverse flower shapes, and long blooming seasons, require annual bulb propagation in horticultural production. This necessity can lead to higher production costs and limit their use in landscaping. The LA hybrid lily 'Aladdin' has shown strong self-reproduction capabilities in optimal cultivation environments, producing numerous high-quality underground stem bulblets. This makes it a valuable model for studying bulblet formation in lilies under natural conditions. Through transcriptome data analysis of different developmental stages of 'Aladdin' bulblets, the LaLBD37 gene, linked to bulblet formation, was identified. Bioinformatics analysis, subcellular localization studies, and transcriptional activation activity tests were conducted to understand the characteristics of LaLBD37. By introducing the LaLBD37 gene into 'Sorbonne' aseptic seedlings via Agrobacterium-mediated transformation, resistant plants were obtained. Positive plants were identified through various methods such as GUS activity detection, PCR, and fluorescence quantitative PCR. Phenotypic changes in positive plants were observed, and various physiological indicators were measured to confirm the role of LaLBD37 in bulblet formation, including soluble sugar content, starch content, sucrose synthase activity, and endogenous hormone levels. The findings suggest that the LaLBD37 gene plays a significant role in promoting the development of lily bulblets, offering insights for enhancing the reproductive capacity of Oriental hybrid lilies and exploring the molecular mechanisms involved in lily bulb regeneration.

Unraveling the Molecular Mechanisms by Which the miR171b-SCL6 Module Regulates Maturation in Lilium.

Lilium is one of the most widely cultivated ornamental bulbous plants in the world. Although research has shown that variable temperature treatments can accelerate the development process from vegetative to reproductive growth in Lilium, the molecular regulation mechanisms of this development are not clear. In this study, Lbr-miR171b and its target gene, LbrSCL6, were selected and validated using transgenic functional verification, subcellular localization, and transcriptional activation. This study also investigated the differential expression of Lbr-miR171b and LbrSCL6 in two temperature treatment groups (25 °C and 15 °C). Lbr-miR171b expression significantly increased after the temperature change, whereas that of LbrSCL6 exhibited the opposite trend. Through in situ hybridization experiments facilitated by the design of hybridization probes targeting LbrSCL6, a reduction in LbrSCL6 expression was detected following variable temperature treatment at 15 °C. The transgenic overexpression of Lbr-miR171b in plants promoted the phase transition, while LbrSCL6 overexpression induced a delay in the phase transition. In addition, LbrWOX4 interacted with LbrSCL6 in yeast two-hybrid and bimolecular fluorescence complementation assays. In conclusion, these results explain the molecular regulatory mechanisms governing the phase transition in Lilium.

GDSL in Lilium pumilum (LpGDSL) Confers Saline-Alkali Resistance to the Plant by Enhancing the Lignin Content and Balancing the ROS.

In order to explore the response mechanism of Lilium pumilum (L. pumilum) to saline-alkali stress, we successfully cloned LpGDSL (GDSL lipase, Gly-Asp-Ser-Leu) from L. pumilum. The qRT-PCR results indicated that the LpGDSL expression was higher in the leaves of L. pumilum, and the expression of the LpGDSL reached the highest level at 12 h in leaves under 11 mM H2O2, 200 mM NaCl, 25 mM Na2CO3, and 20 mM NaHCO3. The bacteriophage overexpressing LpGDSL was more tolerant than the control under different NaHCO3 contents. Overexpressed and wild-type plants were analyzed for phenotype, chlorophyll content, O2- content, H2O2 content, lignin content, and so on. Overexpressed plants had significantly higher resistance than the wild type and were less susceptible to saline-alkali stress. The yeast two-hybrid and BiFC assays demonstrated the existence of an interaction between LpGDSL and LpBCP. The yeast one-hybrid assay and transcriptional activation assay confirmed that B3 transcription factors could act on LpGDSL promoters. Under saline-alkali stress, L. pumilum will promote the expression of LpGDSL, which will then promotes the accumulation of lignin and the scavenging of reactive oxygen species (ROS) to reduce its damage, thus improving the saline-alkali resistance of the plant.

Low LdMYB12 expression contributes to petal spot deficiency in Lilium davidii var. unicolor.

Petal spots are widespread in plants, they are important for attracting pollinators and as economic traits in crop breeding. However, the genetic and developmental control of petal spots has seldom been investigated. To further clarify the development of petal spots formation, we performed comparative transcriptome analysis of Lilium davidii var. unicolor and Lilium davidii petals at the full-bloom stage. In comparison with the parental species L. davidii, petals of the lily variety L. davidii var. unicolor do not have the distinct anthocyanin spots. We show that among 7846 differentially expressed genes detected, LdMYB12 was identified as a candidate gene contributing to spot formation in lily petals. The expression level of LdMYB12 in the petals of L. davidii was higher than that in L. davidii var. unicolor petals. Moreover, overexpression of LdMYB12 led to the appearance of spots on the petals of L. davidii var. unicolor, accompanied by increased expression of anthocyanin synthesis-related genes. Taken together, these results indicate that abnormal expression of LdMYB12 contributes to petal spot deficiency in L. davidii var. unicolor.

Regulatory mechanism of the miR172e-LbrAP2 module during the vegetative growth phase transition in Lilium.

MAIN CONCLUSION: It was proved for the first time that the miR172e-LbrAP2 module regulated the vegetative growth phase transition in Lilium, which provided a new approach to shorten the juvenile stage of Lilium, improved the reproduction rate, and reduced the propagation cost of Lilium commercial bulbs. Lilium is an ornamental bulb plant that takes at least 3 years to cultivate into commercial seed bulbs under natural conditions. The aim of this study was to shorten the Lilium expansion cycle. In this study, the growth cycle of lily tubers induced by low temperature of 15 °C was significantly shorter than that of tubers grown at a conventional temperature. Quantitative real-time PCR analysis showed that the expression patterns of miR172e and LbrAP2 were negatively correlated. GUS histochemical staining confirmed that miR172e and LbrAP2 in tobacco leaves interacted with each other after co-transformation. The shear sites of miR172e and its target gene, LbrAP2, upon binding, were identified by RLM 5' RACE analysis. In addition, miR172e and LbrAP2 showed opposite expression patterns after the transformation of Arabidopsis. miR172e overexpression accelerated the transition from juvenile to adult plants, whereas LbrAP2 overexpression inhibited this process, thus indicating that miR172e negatively regulated the target gene LbrAP2. Upregulation of the transcription factor LbrAP2 delayed the phase transition of plants, whereas miR172 inhibited the transcriptional translation of LbrAP2, thereby accelerating the phase transition. Low-temperature treatment of Lilium bulbs can shorten Lilium development, which provides a new approach to accelerating Lilium commercial bulb breeding and reducing breeding costs.

WUSCHEL-related homeobox genes cooperate with cytokinin to promote bulbil formation in Lilium lancifolium.

The bulbil is an important vegetative reproductive organ in triploid tiger lily (Lilium lancifolium). Based on our previously obtained transcriptome data, we screened two WUSCHEL-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 (Arabidopsis thaliana) 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. Cytokinin type-B response regulators could bind to the promoters of LlWOX9 and LlWOX11 and promote their transcription. LlWOX11 could enhance cytokinin pathway signaling 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.

LiMYB108 is involved in floral monoterpene biosynthesis induced by light intensity in Lilium 'Siberia'.

KEY MESSAGE: We find that the MYB family transcription factor, LiMYB108, has a novel function to regulate the floral fragrance affected by light intensity. Floral fragrance determines the commercial value of flowers and is influenced by many environmental factors, especially light intensity. However, the mechanism by which light intensity affects the release of floral fragrance is unclear. Here, we isolated an R2R3-type MYB transcription factor LiMYB108, the expression of which was induced by light intensity and located in the nucleus. Light of 200 and 600 µmol m-1 s-1 significantly increased the expression of LiMYB108, which was consistent with the improving trend of monoterpene synthesis under light. Virus-induced gene silencing (VIGS) of LiMYB108 in Lilium not only significantly inhibited the synthesis of ocimene and linalool, but also decreased the expression of LoTPS1; however, transient overexpression of LiMYB108 exerted opposite effects. Furthermore, yeast one-hybrid assays, dual-luciferase assays, and electrophoretic mobility shift assays (EMSA) demonstrated that LiMYB108 directly activated the expression of LoTPS1 by binding to the MYB binding site (MBS) (CAGTTG). Our findings demonstrate that light intensity triggered the high expression of LiMYB108, and then LiMYB108 as a transcription factor to activate the expression of LoTPS1, thus promoting the synthesis of the ocimene and linalool, which are important components of floral fragrance. These results provide new insights into the effects of light intensity on floral fragrance synthesis.

A Modified Method for Transient Transformation via Pollen Magnetofection in Lilium Germplasm.

Lily (Lilium spp.) is a popular ornamental plant. Traditional genetic transformation methods have low efficiency in lily, thus development of a high-efficiency genetic transformation system is important. In this study, a novel transient transformation method involving pollen magnetofection was established and optimized pollen viability, and exogenous gene expression in magnetofected pollen and that of different germplasm were assessed. The highest germination percentage of Lilium regale pollen was 85.73% in medium containing 100 g/L sucrose, 61.5 mg/L H3BO3, and 91.5 mg/L CaCl2. A 1:4 ratio of nanomagnetic beads to DNA plasmid and transformation time of 0.5 h realized the highest transformation efficiency (88.32%). The GFP activity in transformed pollen averaged 69.66%, while that of the control pollen was 0.00%. In contrast to the control, transgenic seedlings obtained by pollination with magnetofected pollen showed strong positive GUS activity with 56.34% transformation efficiency. Among the lily germplasm tested, 'Sweet Surrender' and L. leucanthum had the highest transformation efficiency (85.80% and 54.47%), whereas L. davidii var. willmottiae was not successfully transformed. Transformation efficiency was positively correlated with pollen equatorial diameter and negatively correlated with polar axis/equatorial diameter ratio. The results suggest that pollen magnetofection-mediated transformation can be applied in Lilium but might have species or cultivar specificity.

Molecular and Metabolic Insights into Anthocyanin Biosynthesis for Spot Formation on Lilium leichtlinii var. maximowiczii Flower Petals.

Plants exhibit remarkable diversity in their petal colors through biosynthesis and the accumulation of various pigments. Lilium, an important cut and potted flower, has many coloring pattern variations, including bicolors and spots. To elucidate the mechanisms regulating spot formation in Lilium leichtlinii var. maximowiczii petals, we used multiple approaches to investigate the changes in petal carotenoids, spot anthocyanins, and gene expression dynamics. This included green petals without spots (D1-Pe and D1-Sp), yellow-green petals with purple spots (D2-Pe and D2-Sp), light-orange petals with dark-purple spots (D3-Pe and D3-Sp), and orange petals with dark-purple spots (D4-Pe and D4-Sp). D3-Pe and D4-Pe contained large amounts of capsanthin and capsorubin and small amounts of zeaxanthin and violaxanthin, which contributed to the orange color. In addition to cyanidin-3-O-glucoside, pelargonidin-3-O-rutinoside, cyanidin-3-O-rutinoside, and peonidin-3-O-rutinoside may also contribute to L. leichtlinii var. maximowiczii's petal spot colors. KEGs involved in flavonoid biosyntheses, such as CHS, DFR, and MYB12, were significantly upregulated in D2-Sp and D3-Sp, compared with D1-Sp, as well as in spots, compared with petals. Upregulated anthocyanin concentrations and biosynthesis-related genes promoted spot formation and color transition. Our results provide global insight into pigment accumulation and the regulatory mechanisms underlying spot formation during flower development in L. leichtlinii var. maximowiczii.

Distance-based measurement determines the coexistence of B protein hetero- and homodimers in lily tepal and stamen tetrameric complexes.

The floral quartet model proposes that plant MADS box proteins function as higher order tetrameric complexes. However, in planta evidence for MADS box tetramers remains scarce. Here, we applied a strategy using in vivo fluorescence resonance energy transfer (FRET) based on the distance change and distance symmetry of stable tetrameric complexes in tobacco (Nicotiana benthamiana) leaf cells to improve the accuracy of the estimation of heterotetrameric complex formation. This measuring system precisely verified the stable state of Arabidopsis petal (AP3/PI/SEP3/AP1) and stamen (AP3/PI/SEP3/AG) complexes and showed that the lily (Lilium longiflorum) PI co-orthologs LMADS8 and LMADS9 likely formed heterotetrameric petal complexes with Arabidopsis AP3/SEP3/AP1, which rescued petal defects of pi mutants. However, L8/L9 did not form heterotetrameric stamen complexes with Arabidopsis AP3/SEP3/AG to rescue the stamen defects of the pi mutants. Importantly, this system was applied successfully to find complicated tepal and stamen heterotetrameric complexes in lily. We found that heterodimers of B function AP3/PI orthologs (L1/L8) likely coexist with the homodimers of PI orthologs (L8/L8, L9/L9) to form five (two most stable and three stable) tepal- and four (one most stable and three stable) stamen-related heterotetrameric complexes with A/E and C/E function proteins in lily. Among these combinations, L1 preferentially interacted with L8 to form the most stable heterotetrameric complexes, and the importance of the L8/L8 and L9/L9 homodimers in tepal/stamen formation in lily likely decreased to a minor part during evolution. The system provides substantial improvements for successfully estimating the existence of unknown tetrameric complexes in plants.

Lily HD-Zip I Transcription Factor LlHB16 Promotes Thermotolerance by Activating LlHSFA2 and LlMBF1c.

HD-Zip I transcription factors play important roles in plant development and response to abiotic stresses; however, their roles in thermotolerance are largely unknown. Through transcriptome analysis in lily (Lilium longiflorum), we isolated and identified a HD-Zip I gene differentially expressed at high temperatures, LlHB16, which belongs to the ß2 subgroup and positively regulates thermotolerance. The expression of LlHB16 was rapidly and continuously activated by heat stress. LlHB16 protein localized to the nucleus and exhibited transactivation activity in both plant and yeast cells, and its C-terminus contributed to its transcriptional activity. Overexpressing LlHB16 in Arabidopsis and lily improved thermotolerance and activated the expression of heat-related genes in both plants, especially that of HSFA2 and MBF1c. In addition, LlHB16 overexpression in Arabidopsis also caused growth defects, delayed flowering and abscisic acid (ABA) insensitivity. Further analysis revealed that LlHB16 directly binds to the promoters of LlHSFA2 and LlMBF1c and activates their expressions. Similarly, the expression of AtHSFA2 and AtMBF1c was also elevated in LlHB16 transgenic Arabidopsis lines. Together, our findings demonstrate that LlHB16 participates in the establishment of thermotolerance involved in activating LlHSFA2 and LlMBF1c, and LlHB16 overexpression resulted in ABA insensitivity in transgenic plants, suggesting that LlHB16 links the basal heat-responsive pathway and ABA signal to collaboratively regulate thermotolerance.

Small HSPs play an important role in crosstalk between HSF-HSP and ROS pathways in heat stress response through transcriptomic analysis in lilies (Lilium longiflorum).

BACKGROUND: High temperature seriously limits the annual production of fresh cut lilies, which is one of the four major cut flowers in the global cut flower market. There were few transcriptomes focused on the gene expression of lilies under heat stress. In order to reveal the potential heat response patterns in bulbous plants and provide important genes for further genetic engineering techniques to improve thermotolerance of lily, RNA sequencing of lilies under heat treatments were conducted. RESULTS: In this study, seedlings of Lilium longiflorum 'White Heaven' were heat-treated at 37 °C for different lengths of time (0 h, 0.5 h, 1 h, 3 h, 6 h, and 12 h with a 12 h-light/12 h-dark cycle). The leaves of these lily seedlings were immediately collected after heat treatments and quickly put into liquid nitrogen for RNA sequencing. 109,364,486-171,487,430 clean reads and 55,044 unigenes including 21,608 differentially expressed genes (DEGs) (fold change ≥2) were obtained after heat treatment. The number of DEGs increased sharply during the heat treatments of 0.5 h-1 h and 1 h-3 h compared to that of other periods. Genes of the heat stress transcription factor (HSF) family and the small heat shock proteins (small HSPs, also known as HSP20) family responded to heat stress early and quickly. Compared to that of the calcium signal and hormone pathways, DEGs of the HSF-HSP pathway and reactive oxygen species (ROS) pathway were significantly and highly induced. Moreover, they had the similar expression pattern in response to heat stress. Small HSPs family genes were the major components in the 50 most highly induced genes at each heat stress treatment and involved in ROS pathway in the rapid response to heat stress. Furthermore, the barley stripe mosaic virus induced gene silencing (BSMV-VIGS) of LlHsfA2 caused a significantly reduced thermotolerance phenotype in Lilium longiflorum 'White Heaven', meanwhile decreasing the expression of small HSPs family genes and increasing the ROS scavenging enzyme ascorbate peroxidase (APX) genes, indicating the potential interplay between these two pathways. CONCLUSIONS: Based on our transcriptomic analysis, we provide a new finding that small HSPs play important roles in crosstalk between HSF-HSP and ROS pathways in heat stress response of lily, which also supply the groundwork for understanding the mechanism of heat stress in bulbous plants.

Comparative metabolome analysis unravels a close association between dormancy release and metabolic alteration induced by low temperature in lily bulbs.

KEY MESSAGE: The correlation between dormancy release and metabolic metabolic changes in lily bulbs during low temperature storage was investigated. Low temperature is a major environmental factor required for dormancy release in lily bulbs. Although great advances in plant metabolomics have been achieved, knowledge about the molecular basis of lily bulb metabolomes at different developmental stages in response to low temperature is still limited. In this work, the dormancy release, vegetative growth, flowering, metabolic profile and gene expression in the less dormant cultivar Lilium longiforum × Oriental hybrid 'Triumphator' (T) and the more dormant cultivar Lilium Asiatic hybrid 'Honesty' (H) were compared. Exposure to low temperature (LT) successfully promoted stem elongation, floral transition and flowering of both T and H bulbs. However, exposure to room temperature (RT) restricted stalk elongation of both T and H bulbs, and prohibited floral transition and flowering of H bulbs. Correspondingly, higher antioxidant enzyme activity and total primary metabolite contents were observed in the apical bud of T bulbs. Gene expression analysis revealed that expressions of LiFT, LiFLK, LiSOC1 and LiCBF were decreased, whereas the expression of LiSVP and LiFLC were increased, in the apical bud of H bulbs under RT storage condition. Our findings reveal that the growth and dormancy breaking of lily bulbs are closely associated with the metabolic changes in the apical buds during postharvest storage.

Analyses of Botrytis cinerea-responsive LrWRKY genes from Lilium regale reveal distinct roles of two LrWRKY transcription factors in mediating responses to B. cinerea.

KEY MESSAGE: Botrytis cinerea induced expression of 15 LrWRKY genes; overexpression of LrWRKY39 and LrWRKY41a increased resistance and susceptibility, respectively, to B. cinerea in a manner related to SA and JA signaling. WRKY transcription factors (TFs), a large family, play important roles in coping with biotic stresses. Lilium regale Wilson is a lily species with strong resistance to fungi and viruses; however, functional characterization of LrWRKY TFs remains very limited. Here, a total of 25 LrWRKY members were identified from the L. regale transcriptome, and 15 LrWRKY genes were significantly induced by Botrytis cinerea. Based on their structural features, B. cinerea-responsive LrWRKY genes could be classified into six subgroups (Groups I, IIa-d, and III), and sequence alignment showed that 12 LrWRKY proteins have a well-conserved WRKYGQK domain, while 3 LrWRKYs have a variant sequence (WRKYGKK or WRMYEQK). Quantitative RT-PCR analysis revealed tissue-specific expression of B. cinerea-responsive LrWRKY genes and their expression profiles in response to defense-related hormones salicylic acid (SA), methyl jasmonate (MeJA) and hydrogen peroxide. LrWRKY39 and LrWRKY41a, which encode two LrWRKY TFs with different three-dimensional (3D) models of the WRKY domain, were cloned, and both proteins were targeted to the nucleus. Overexpression of LrWRKY39 and LrWRKY41a in Arabidopsis thaliana increased the resistance and susceptibility to B. cinerea, respectively, compared to the wild type. Similar results were also observed in tobacco and lily (L. longiflorum 'Snow Queen') by transient transformation analyses. Their distinct roles may be related to changes in the transcript levels of SA-/JA-responsive genes. Our results provide new insights into B. cinerea-responsive LrWRKY members and the biological functions of two different 3D models of LrWRKY TFs in defense responses to B. cinerea infection.

Full-Length Transcriptome Characterization and Functional Analysis of Pathogenesis-Related Proteins in Lilium Oriental Hybrid 'Sorbonne' Infected with Botrytis elliptica.

Gray mold (Botrytis elliptica) causes a deleterious fungal disease that decreases the ornamental value and yield of lilies. Lilium oriental hybrid 'Sorbonne' is a variety that is resistant to gray mold. Understanding the mechanism of resistance against B. elliptica infection in 'Sorbonne' can provide a basis for the genetic improvement in lily plants. In this study, a PacBio Sequel II system was used to sequence the full-length transcriptome of Lilium 'Sorbonne' after inoculation with B. elliptica. A total of 46.64 Gb subreads and 19,102 isoforms with an average length of 1598 bp were obtained. A prediction analysis revealed 263 lncRNAs, and 805 transcription factors, 4478 simple sequence repeats, and 17,752 coding sequences were identified. Pathogenesis-related proteins (PR), which may play important roles in resistance against B. elliptica infection, were identified based on the full-length transcriptome data and previously obtained second-generation transcriptome data. Nine non-redundant potential LhSorPR proteins were identified and assigned to two groups that were composed of two LhSorPR4 and seven LhSorPR10 proteins based on their genetic relatedness. The real-time quantitative reverse transcription PCR (qRT-PCR) results showed that the patterns of expression of nine differentially expressed PR genes under B. elliptica stress were basically consistent with the results of transcriptome sequencing. The pattern of expression of LhSorPR4s and LhSorPR10s genes in different tissues was analyzed, and the expression of each gene varied. Furthermore, we verified the function of LhSorPR4-2 gene in Lilium. The expression of LhSorPR4-2 was induced by phytohormones such as methyl jasmonate, salicylic acid, and ethephon. Moreover, the promoter region of LhSorPR4-2 was characterized by several functional domains associated with phytohormones and stress response. The overexpression of LhSorPR4-2 gene in 'Sorbonne' increased the resistance of the lily plant to B. elliptica and correlated with high chitinase activity. This study provides a full-length transcript database and functionally analyzed the resistance of PR gene to B. elliptica in Lilium, thereby introducing the candidate gene LhSorPR4-2 to breed resistance in Lilium.

Expression Patterns of Key Genes in the Photoperiod and Vernalization Flowering Pathways in Lilium longiflorum with Different Bulb Sizes.

Lilium longiflorum is a wild Lilium, and its flowering transition requires a long period of cold exposure to meet the demand of vernalization. The responses of different sized bulbs to cold exposure and photoperiod are different, and the floral transition pathways of small and large bulbs are different. In this study, small and large bulbs were placed in cold storage for different weeks and then cultured at a constant ambient temperature of 25 °C under long day (LD) and short day (SD) conditions. Then, the flowering characteristics and expression patterns of key genes related to the vernalization and photoperiod pathways in different groups were calculated and analyzed. The results showed that the floral transition of Lilium longiflorum was influenced by both vernalization and photoperiod, that vernalization and LD conditions can significantly improve the flowering rate of Lilium longiflorum, and that the time from planting to visible flowering buds' appearance was decreased. The flowering time and rate of large bulbs were greatly influenced by cold exposure, and the vernalization pathway acted more actively at the floral transition stage. The floral transition of small bulbs was affected more by the photoperiod pathway. Moreover, it was speculated that cold exposure may promote greater sensitivity of the small bulbs to LD conditions. In addition, the expression of LlVRN1, LlFKF1, LlGI, LlCO5, LlCO7, LlCO16, LlFT1, LlFT3 and LlSOC1 was high during the process of floral transition, and LlCO13, LlCO14 and LlCO15 were highly expressed in the vegetative stage. The expression of LlCO13 and LlCO14 was different under different lighting conditions, and the flowering induction function of LlCO9 and LlFT3 was related to vernalization. Moreover, LlFKF1, LlGI, LlCO5, LlCO16, LlSOC1 and LlFT2 were involved in the entire growth process of plants, while LlCO6, LlCO16 and LlFT1 are involved in the differentiation and formation of small bulblets of plants after the inflorescence stage, and this process is also closely related to LD conditions. This study has great significance for understanding the molecular mechanisms of the vernalization and photoperiod flowering pathways of Lilium longiflorum.