WRKY genes represent a major family of transcription factors that play key roles in regulating plant responses to both biotic and abiotic stresses. While WRKY genes have been extensively studied in various plant species, their functions in lily remain largely unknown. Here, we identified 115 WRKY family genes in the lily genome through bioinformatics analyses. These genes encode proteins ranging from 72 to 708 amino acids, with predicted isoelectric points between 4.90 and 11.06. Phylogenetic analysis grouped these LdWRKY genes into clusters I, II, and III, and promoter analysis identified numerous cis-acting elements related to growth, development, and hormone and stress responses. Gene expression heatmaps revealed significant variation in the expression of LdWRKY family members across different tissues, with 62 out of 115 LdWRKYs showing differential expression under heat stress conditions. Using transcriptome data, we cloned the heat stress-responsive candidate gene LdWRKY87, which encodes a nuclear-localized protein with transcriptional repression activity. Transient transformation assays confirmed the role of LdWRKY87 in regulating lily heat tolerance. Further studies suggest that LdWRKY87 may modulate heat tolerance by affecting the expression of heat tolerance-related genes, such as LdHSFA2, LdHSP70, and LdMBF1c. These findings provide a foundation for future studies on the regulatory mechanisms of WRKY transcription factors in lily heat tolerance.

BBX (B-box) proteins are critical regulators in plant growth and development, playing pivotal roles in processes such as photomorphogenesis and floral transition. Additionally, they are involved in plant responses to various abiotic stresses, including salt, drought, and cold. However, their specific roles in thermotolerance remain largely unexplored. In this study, we identified a heat-inducible BBX gene, LlBBX15, which belongs to the class III BBX subfamily of lily (Lilium longiflorum). LlBBX15 localized to the nucleus and exhibited transcriptional activation activity. Stable overexpression of LlBBX15 resulted in increased thermotolerance in both Arabidopsis (Arabidopsis thaliana) and lily, whereas silencing LlBBX15 in lily led to a reduction in thermotolerance. Furthermore, LlBBX15 interacted with LlbHLH87 (BASIC HELIX-LOOP-HELIX FACTOR 87) and directly bound to the promoter of LlHSFA2 (HEAT STRESS TRANSCRIPTION FACTOR A2), thereby activating its expression. Subsequent analyses revealed that the heterologous interaction between LlBBX15 and LlbHLH87 facilitated their respective homologous interactions. A complex of LlBBX15 and LlbHLH87 enhanced their DNA-binding capacity and cooperatively promoted the expression of LlHSFA2. Moreover, both LlHSFA1 and LlHSFA2 were identified as direct regulators of LlBBX15, with evidence suggesting a synergetic activation effect on its expression. This interaction indicates the existence of a feedback loop between the HSFs and LlBBX15. Collectively, these findings establish LlBBX15 as a positive regulator that collaborates with LlbHLH87 within the HSF signaling pathway to facilitate thermotolerance in plants.

The serine/threonine protein kinase LlSAPK2 enhances thermotolerance in lily (Lilium longiflorum) by phosphorylating and stabilizing the transcription factor LlbZIP46, which activates heat shock factor genes, thereby reducing heat-induced cellular damage.

Heat stress significantly damages crop yield and quality. PLATZ (PLANT A/T-RICH SEQUENCE-AND ZINC-BINDING PROTEIN) transcription factors play pivotal roles in plant growth, development, and environmental stress responses. While the functions of PLATZ members in response to drought and salt stress are well characterised, their roles in heat stress remain largely unexplored. Here, LlPLATZ1, a heat-inducible member of the PLATZ family from lily (Lilium longiflorum), was identified. LlPLATZ1 was rapidly induced by high temperature, and its protein was localised to the nucleus, showing transcriptional repression activity. LlPLATZ1 bound to the promoter of a class B heat stress transcription factor gene, LlHSF24, to inhibit its expression. Stable overexpression of LlPLATZ1 in lily enhanced its thermotolerance, whereas silencing LlPLATZ1 had the opposite effect. Further analysis showed that LlHSF24 directly repressed the expression of heat-protective genes LlHSP22.0 and LlHSP70 to weaken thermotolerance. In addition, LlPLATZ1 interacted with LlMYB4, a later heat-inducible MYB transcription factor that bound to the LlHSF24 promoter to activate its expression. LlMYB4 limited the heat stress response by interacting with LlPLATZ1 to antagonise its DNA-binding ability. In combination, these results indicate that the LlPLATZ1/LlMYB4-LlHSF24 module may play a crucial role in maintaining a balanced heat stress response, enabling plants to adapt to complex environmental changes.

Abstract Bulblet initiation and expansion are critical developmental phases in lily, which were actively regulated by hormone and carbohydrate metabolism. However, the integrative molecular mechanisms linking these pathways remain poorly understood. Here, we delineated nine major cell layers during bulblets formation using spatial transcriptome and found their transcriptional programs to be enriched in hormone and carbohydrate metabolism pathways. We demonstrated that cytokinin accelerates sucrose degradation during bulblet regeneration stage and promotes sucrose and starch synthesis during bulblet expansion. Two type-B response regulators (RRs), LiRR12 and LiRR14, were characterized as key positive regulators of this process. Silencing LiRR12 and LiRR14 suppressed bulblet regeneration and expansion, while transient overexpression in lily and heterologous expression in Arabidopsis promoted plant aboveground growth. Further evidences indicated that LiRR14 transcriptionally activates LiCWIN4 to accelerate sucrose hydrolysis, thereby promoting bulblet regeneration. Subsequently, LiRR12 and LiRR14 activate LiSPS3 and LiSSS1, respectively, to enhance sucrose biosynthesis and starch accumulation, ultimately promoting bulblet expansion. Collectively, our findings establish LiRR12 and LiRR14 as central regulators that integrate cytokinin signaling with stage-specific carbohydrate metabolism to organize lily bulblet development.

Heat stress (HS) is a major environmental stress that inhibits plant growth and development. Plants have evolved various mechanisms to cope with heat stress, a key one being the HSF-HSP (Heat stress transcription factor-Heat shock protein) signaling pathway. HSFs can be divided into three classes: A, B, and C. In this study, we report the identification and functional characterization of a specific B2 member LdHSFB2a in Lilium davidii var. unicolor. RT-qPCR (Real-time Quantitative Polymerase Chain Reaction) analyses indicated that LdHSFB2a was highly expressed in HS-exposed leaves. LdHSFB2a was localized in the nucleus, consistent with the characterization of transcription factors. In contrast to other HSFBs, LdHSFB2a did not contain the typical B3 repression domain but exhibited transcriptional repression activity in yeast and plant cells. Transient overexpression and virus-induced gene silencing (VIGS) of LdHSFB2a in lily petals suggested that LdHSFB2a functions positively in lily thermotolerance. Consistent with the implication of LdHSFB2a function in thermotolerance, further analysis revealed that the expression levels of HSFA1, HSFA2, and MBF1c were increased as LdHSFB2a was overexpressed but reduced as LdHSFB2a was silenced. Furthermore, LdHSFB2a bound to the promoters of HSFA3 A, WRKY33, CAT2, and GLOS1. And LdHSFB2a overexpression and silencing enhanced and reduced their expressions, respectively. Therefore, we speculated that LdHSFB2a may be a coactivator that interacts with transcriptional activators to promote thermotolerance in lily by enhancing the expression of heat-responsive genes such as HSFA3 A, WRKY33, CAT2, and GLOS1.

INTRODUCTION:Timely anther dehiscence is a key step for successful sexual reproduction in plants. Secondary cell wall thickening of anther endothecium is a vital process during anther dehiscence that provides an indispensable mechanical force for successful dehiscence. Anther dehiscence depends on anther lignification, and it is a timely and sophisticated process regulated by phytohormones and transcription factors. However, whether endothecium lignification occurs during anther dehiscence in lily and underlying mechanisms are still largely unclear. OBJECTIVES:Our work focuses on identifying the course of endothecium lignification during anther dehiscence and elucidating the molecular mechanisms underlying endothecium lignification-dependent anther dehiscence in lily. METHODS:Lignin fluorescence analysis and ultraviolet spectrophotometry were employed to elucidate the endothecium lignification process. Target genes were isolated from the transcriptomic data of anther dehiscence and lignification process. Virus-induced gene silencing (VIGS) and transient overexpression in lily anthers were used to analyze the LoMYB26 function. Yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), and dual-luciferase (LUC) assay analyzed the regulatory mechanisms. Yeast two-hybrid (Y2H), luciferase complementation imaging (LCI), and bimolecular fluorescence complementation (BiFC) assays illustrated the interaction between LoMYB26 and LoJAZ4. RESULTS:Our results showed that endothecium lignification occurred in S6-S7 stages when anther dehiscence had not yet occurred. The R2R3-type MYB transcription factor, LoMYB26, was found to promote endothecium lignification. LoMYB26 directly bound to the Caffeic Acid O-methyltransferase (LoCOMT) promoter and activated its transcription. Meanwhile, LoMYB26 interacted with jasmonate-ZIM domain protein 4 (LoJAZ4), which repressed the LoMYB26-mediated activation of LoCOMT transcription. Additionally, the exogenous application of methyl-jasmonate (Me-JA) induced LoMYB26 transcription and promoted endothecium lignification. CONCLUSION:Our findings demonstrate that LoMYB26 promotes endothecium lignification and anther dehiscence. LoMYB26 interacted with LoJAZ4, forming a heterodimer that participates in JA-mediated endothecium lignification and anther dehiscence. This study offers valuable insights and a theoretical foundation for the breeding of anther-indehiscent lily.

Lilium holds significant horticultural and ecological importance. Understanding the morpho-anatomical diversity of the stems can provide insights into taxonomy and breeding strategies. This study comprehensively examined the stem morpho-anatomy of 71 Lilium taxa to elucidate taxonomic and structural differences. For the first time, four distinct jigsaw-puzzle-shaped shapes of epidermal cells (Ep) in monocot stems, novel I-shaped and Co-xylem (O-, X-, W-, Q-shaped) vascular bundles (Vb) in Lilium stems, and quantitative characteristics (Vb density, xylem/phloem area ratio, etc.) were systematically discovered and analyzed. Asiatic (A) and Longiflorum × A (LA) hybrids displayed epidermal appendages, while Oritenal × Trumpet (OT) hybrids featured thicker sclerenchymatous rings (Sr). Collateral Vb in hybrids visually displayed bicollateral with degraded bundle sheaths (Bs), contrasting with intact circular Bs in wild species. Ward.D clustering categorized Lilium taxa into group A (Oritenal and OT hybrids) and B (A, LA, Trumpet, Longiflorum × Oriental hybrids and wild species), with Mantel’s test identified height, Ep shape, Ep length/width ratio, cortex/Sr thickness ratio and Bs integrity as key discriminators. Bending stems exhibited a higher Vb area. These findings establish a comprehensive pheno-anatomical framework for Lilium, which can guide future breeding programs and ecological studies.

Multiprotein bridging factor 1c (MBF1c) has been shown to play a critical role in plant responses to heat stress. Previous studies have implicated MBF1c roles in ethylene-mediated thermotolerance; however, the upstream regulatory mechanisms linking MBF1c to this process remain unclear. In this study, an ethylene-response factor (ERF), LlERF092, was identified as a potential regulator of LlMBF1c through a yeast one-hybrid screening assay. Further investigations revealed that LlERF092 directly bound to the promoter of LlMBF1c and activated its transcription. LlERF092 was rapidly induced by heat stress, and its protein localised to the nucleus. Overexpression of LlERF092 enhanced the thermotolerance of the transgenic lily plants. Furthermore, immunoprecipitation followed by mass spectrometry (IP-MS) identified LlETO1 (ETHYLENE OVERPRODUCER 1) as an interacting partner of LlERF092. The expression of LlETO1 was activated in response to transient heat stress, and the LlETO1-LlERF092 interaction enhanced the transcriptional activity of LlERF092. Co-overexpression of LlERF092 and LlETO1 enhanced thermotolerance more than the overexpression of either gene alone, while co-silencing of LlERF092 and LlETO1 further reduced thermotolerance compared to silencing each gene individually. Additionally, heat stress promoted ethylene production in lily leaves, and exogenous application of ethephon enhanced thermotolerance. Ethephon treatment also elevated the expression of LlERF092, LlETO1, and LlMBF1c, while their expression was repressed by 1-MCP under heat stress. In summary, these findings demonstrated that the LlERF092/LlETO1-LlMBF1c transcriptional cascade mediated ethylene-dependent thermotolerance in lily under heat stress conditions. This study provides new insights into the molecular mechanisms underlying plant heat stress responses and highlights the role of ethylene signalling in thermotolerance.

Heat stress transcription factors (HSFs) are core factors of plants in response to heat stress (HS), but their regulatory network is complicated and remains elusive in a large part, especially HSFBs. In this study, we reported that the LlERF012-LlHSFA1 module participates in heat stress response (HSR) by directly regulating HSF pathway in lily (Lilium longiflorum). LlHSFB1 was confirmed as a positive regulator in lily thermotolerance and a heat-inducible AP2/ERF member LlERF012 (Ethylene Response Factor 012) was further identified to be a direct trans-activator of LlHSFB1. Overexpression of LlERF012 elevated the thermotolerance of transgenic Arabidopsis and lily, but silencing LlERF012 reduced thermotolerance in lily. Further analysis showed LlERF012 interacted with LlHSFA1, which led to enhanced transactivation activity and DNA-binding capability of LlERF012. In addition, LlERF012 also directly activated the expression of LlHSFA1 by binding its promoter. As expected, we found that LlERF012 bound the promoters of LlHSFA2, LlHSFA3A, and LlHSFA3B to stimulate their expression, and LlERF012-LlHSFA1 interaction enhanced these activation effects. Overall, our data suggested that LlERF012 was a key factor for lily thermotolerance and the LlERF012-LlHSFA1 interaction synergistically regulated the activity of the HSF pathway including the class A and B members, which might be of great significance for coordinating the functions of different HSFs.

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.

Basic helix-loop-helix (bHLH) proteins comprise one of the largest families of transcription factors in plants, which play roles in plant development, secondary metabolism, and the response to biotic/abiotic stresses. However, the roles of bHLH proteins in thermotolerance are largely unknown. Herein, we identified a heat-inducible member of the bHLH family in lily (Lilium longiflorum), named LlbHLH87, which plays a role in thermotolerance. LlbHLH87 was rapidly induced by transient heat stress, and its encoded protein was localized to the nucleus, exhibiting transactivation activity in both yeast and plant cells. Overexpression of LlbHLH87 in Arabidopsis enhanced basal thermotolerance, while silencing of LlbHLH87 in lily reduced basal thermotolerance. Further analysis showed that LlbHLH87 bound to the promoters of HEAT STRESS TRANSCRIPTION FACTOR A2 (LlHSFA2) and ETHYLENE-INSENSITIVE 3 (LlEIN3) to directly activate their expression. In addition, LlbHLH87 interacted with itself and with SPATULA (LlSPT) protein. LlSPT was activated by extended heat stress and its protein competed for the homologous interaction of LlbHLH87, which reduced the transactivation ability of LlbHLH87 for target genes. Compared with that observed under LlbHLH87 overexpression alone, co-overexpression of LlbHLH87 and LlSPT reduced the basal thermotolerance of lily to sudden heat shock, but improved its thermosensitivity to prolonged heat stress treatment. Overall, our data demonstrated that LlbHLH87 regulates thermotolerance via activation of LlEIN3 and LlHSFA2, along with an antagonistic interaction with LlSPT.

Homeodomain-leucine zipper (HD-Zip) I transcription factors are crucial for plant responses to drought, salt, and cold stresses. However, how they are associated with thermotolerance remains mostly unknown. We previously demonstrated that lily (Lilium longiflorum) LlHB16 (HOMEOBOX PROTEIN 16) promotes thermotolerance, whereas the roles of other HD-Zip I members are still unclear. Here, we conducted a transcriptomic analysis and identified a heat-responsive HD-Zip I gene, LlHOX6 (HOMEOBOX 6). We showed that LlHOX6 represses the establishment of basal thermotolerance in lily. LlHOX6 expression was rapidly activated by high temperature, and its protein localized to the nucleus. Heterologous expression of LlHOX6 in Arabidopsis (Arabidopsis thaliana) and overexpression in lily reduced their basal thermotolerance. In contrast, silencing LlHOX6 in lily elevated basal thermotolerance. Cooverexpressing or cosilencing LlHOX6 and LlHB16 in vivo compromised their functions in modulating basal thermotolerance. LlHOX6 interacted with itself and with LlHB16, although heterologous interactions were stronger than homologous ones. Notably, LlHOX6 directly bounds DNA elements to repress the expression of the LlHB16 target genes LlHSFA2 (HEAT STRESS TRANSCRIPTION FACTOR A2) and LlMBF1c (MULTIPROTEIN BRIDGING FACTOR 1C). Moreover, LlHB16 activated itself to form a positive feedback loop, while LlHOX6 repressed LlHB16 expression. The LlHOX6-LlHB16 heterooligomers exhibited stronger DNA binding to compete for LlHB16 homooligomers, thus weakening the transactivation ability of LlHB16 for LlHSFA2 and LlMBF1c and reducing its autoactivation. Altogether, our findings demonstrate that LlHOX6 interacts with LlHB16 to limit its transactivation, thereby impairing heat stress responses in lily.

Lily (Lilium spp.) is popular for its colorful flowers and exquisite scents. Nonetheless, high temperatures often severely reduce its yield production and quality. The implementation of biotechnological approaches to manipulate the expression of key heat-resistant genes is an effective way to improve the thermotolerance of plants. Here, we isolated a gene encoding for a multi-protein bridging factor 1c (MBF1c) from L. longiflorum 'White Heaven' (LlMBF1c), which was highly similar to MBF1c from Elaeis guineensis (EgMBF1c). LlMBF1c harbors conserved MBF1 and helix-turn-helix (HTH) domains. Moreover, the expression of LlMBF1c and its promoter activity were enhanced under high-temperature conditions. Further analysis indicated that LlMBF1c is a transcriptional repressor in both yeast and Nicotiana benthamiana. Its protein was located in the nucleus and cytoplasm of N. benthamiana leaf cells. Overexpression of LlMBF1c in lily and Arabidopsis resulted in enhanced thermotolerance in these plants. By contrast, silencing LlMBF1c reduced the thermotolerance of lily. Our results identified an important candidate gene that can be utilized to develop thermotolerant lily germplasm.

Gray mold caused by Botrytis cinerea is one of the major threats in lily production. However, limited information is available about the underlying defense mechanism against B. cinerea in lily. Here, we characterized a nuclear-localized class A heat stress transcription factor (HSF)-LlHSFA4 from lily (Lilium longiflorum), which positively regulated the response to B. cinerea infection. LlHSFA4 transcript and its promoter activity were increased by B. cinerea infection in lily, indicating its involvement in the response to B. cinerea. Virus-induced gene silencing (VIGS) of LlHSFA4 impaired the resistance of lily to B. cinerea. Consistent with its role in lily, overexpression of LlHSFA4 in Arabidopsis (Arabidopsis thaliana) enhanced the resistance of transgenic Arabidopsis to B. cinerea infection. Further analysis showed that LlWRKY33 directly activated LlHSFA4 expression. We also found that both LlHSFA4 and LlWRKY33 positively regulated plant response to B. cinerea through reducing cell death and H2O2 accumulation and activating the expression of the reactive oxygen species (ROS) scavenging enzyme gene LlCAT2 (Catalase 2) by binding its prompter, which might contribute to reducing H2O2 accumulation in the infected area. Taken together, our data suggested that there may be a LlWRKY33-LlHSFA4-LlCAT2 regulatory module which confers B. cinerea resistance via reducing cell death and the ROS accumulation.

The homeostasis of gibberellin (GA) is crucial for the normal development of anthers, but its underlying regulatory mechanisms are not clear. The GA-induced v-Myb myeloblastosis viral oncogene homolog (MYB) transcription factor LoMYB65 is involved in anther development. In this study, we screened and identified an interacting protein of LoMYB65, Lilium Oriental Hybrids BEL1-Like Homeodomain6 (LoBLH6). LoBLH6 was localized in both the nucleus and cytoplasm, and it interacted with LoMYB65 through its BELL domain, exhibiting transcriptional repression activity. LoBLH6 was continuously expressed during anther development, with particularly high expression in the mid and late stages. In situ hybridization revealed high expression of LoBLH6 in the tapetum and microspores, with the same tissue specificity as LoMYB65. Silencing of LoBLH6 in lilies resulted in abnormal anther development, reduced pollen, and increased GA content. The application of GA-induced phenotypes in the anthers and pollen of lily that were similar to the silencing of LoBLH6. Further research showed that LoBLH6 directly binds to the promoter of Lilium Oriental Hybrids GA 20-oxidase1 (LoGA20ox1) to suppress its expression, and coexpression with LoMYB65 enhances this repression. Additionally, GA treatment enhanced the interaction between LoBLH6 and LoMYB65 and their complex's inhibitory effect on downstream target genes. During the transition from microspores to mature pollen grains in lily anthers, GA levels maintain a steady state, which is disrupted by silencing LoBLH6, leading to abnormal pollen development. Overall, our results reveal that the interaction between LoBLH6 and LoMYB65 regulates anther development through feedback regulation of GA synthesis.

Lily（Lilium spp.） is an important horticultural crop, but its use is limited due to serious pollen contamination problems. There are many studies on pollen development in model plants, but few on flower crops such as lilies. Gibberellin（GA） is a large class of hormones and plays an important role in plant vegetative growth and reproductive development. GAMYB is a group of the R2R3-MYB family upregulated by gibberellin,and plays an important role in anther development. Here, we isolated a novel GAMYB, named LoMYB65, from lily, which was closely related to the AtMYB65 and AtMYB33 in Arabidopsis. Fluorescence quantitative PCR results showed that LoMYB65 was mainly expressed in lily anthers.LoMYB65 could be activated by 288 μmol · L-1GA3treatment and the LoMYB65 protein was located in the nucleus and cytoplasm, and had transactivation in yeast and tobacco leaf cells. The conserved motif within 226 amino acids of the C-terminal of LoMYB65 contributed to its transactivation. Overexpression of LoMYB65 caused dwarf phenotype, unnormal tapetum development, less seeds of siliques in transgenic Arabidopsis plants, the transgenic plants showed partly male sterile. Simultaneously, silencing of LoMYB65 with VIGS（Virus Induced Gene Silencing） in lily anthers caused unnormal pollen development and reduced the pollen amount. Overexpression of LoMYB65 in Arabidopsis and silencing of LoMYB65 in lily resulted in decreased pollen counts, so we speculate that LoMYB65 may be dose-dependent. Overall, these findings suggest that LoMYB65 may play an important role in anther development and pollen formation in lily. LoMYB65 may provide a useful candidate gene for pollenless breeding of lily.