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Polyploid plants often show improved resistance against many diseases, but whether they show increased resistance to grey mould, a devastating disease caused by Botrytis spp. fungi, is seldom reported. Stomata and reactive oxygen species (ROS) play dual roles in defence against grey mould, and it is unclear how their roles change after polyploidization. We addressed these questions in diploid and colchicine-induced Lilium rosthornii after B. elliptica infection. Tetraploids were less susceptible to grey mould, with lower morality rates in naturally infected plants. Before the stomata closed in artificially infected leaves, tetraploids, with larger stomatal apertures, were more easily invaded by the pathogen than diploids. However, the lesion area increased more slowly in tetraploids than in diploids, which may be explained by three causes based on histological and physiological characteristics. First, the pathogen required more time to penetrate the epidermis and closed stomata in tetraploids than in diploids. Second, the pathogen penetrated the reopened stomata more easily than the epidermis, and stomatal density was lower in tetraploids than in diploids. Third, tetraploids showed faster ROS accumulation, a more effective ROS-scavenging system and less malondialdehyde (MDA) accumulation than diploids. Stomatal starch and abnormal guard cell nuclei were present in the infected leaves. This phenomenon may be caused by oxalic acid, a pathogenic factor for many pathogens that promotes stomatal starch degradation and stomatal reopening in Sclerotinia spp., a pathogen closely related to Botrytis spp. This suggestion was primarily confirmed by immersing healthy leaves in oxalic acid solution.

Gray mold disease, mainly caused by Botrytis elliptica (Berk.) Cooke and Botrytis cinerea Pers., leads to severe losses in lily cut flower and bulb production. MYC2 is a critical regulator of the activation of jasmonate-mediated defense responses in plants; however, information about the lily MYC2 gene is limited. Therefore, functional characterization of MYC2 in lily, especially its role in plant immune responses, should be performed. Here, significant differences between the Botrytis-resistant Lilium hybrid ‘Sorbonne’ and the Botrytis-susceptible ‘Tresor’ were found following B. cinerea inoculation, as indicated by jasmonic acid (JA) and JA-isoleucine (JA-Ile) accumulation and related gene expression. More JA and JA-Ile were detected in ‘Sorbonne’ than in ‘Tresor’ following fungal inoculation, and higher transcript levels of JA biosynthesis genes (LhAOS, LhAOC, and LhOPR3) and a signaling gene (LhCOI1) were detected in ‘Sorbonne’ than in ‘Tresor’. In contrast, expression of the critical signaling regulator LhMYC2 was higher in ‘Tresor’ than in ‘Sorbonne’. LhMYC2 was then isolated from ‘Sorbonne’ and found to be similar to several plant MYC2 homologs that have pivotal roles in JA signaling. The expression of LhMYC2 increased significantly in response to JA and salicylic acid (SA) in ‘Sorbonne’. Ectopic expression of LhMYC2 in Arabidopsis resulted in greater susceptibility to B. cinerea than that observed in wild-type plants. This susceptibility was coupled with the transcriptional changes in SA- and JA-responsive genes. Overall, our findings indicate that LhMYC2 plays a negative role in Arabidopsis resistance to B. cinerea.

Most recent studies on Lilium species have been descriptive observations based on FISH (fluorescence in situ hybridization) analyses or phylogenetic studies focusing on only one section without taking the evolutionary framework of Lilium into account. To better understand the taxonomic status, evolution, and karyotype diversity of the genus Lilium, we conducted FISH mapping using 35S rDNA sequences in 29 important wild Lilium species covering four sections of the genus in China. Their molecular phylogenetic relationships were analyzed based on the internal transcribed spacer (ITS) region. For 35S rDNA, the hybridization loci always existed in pairs on homologous chromosomes. The number of hybridization signals varied from 4 to 14 in diploid species, and the signals were mainly located on the proximal region of the short arm of chromosomes. Among all the sections, sect. Sinomartagon had the most 35S rDNA signals. Genotype grouping based on 35S signal loci was largely consistent with groups in the phylogenetic tree constructed using ITS sequences, and different species in a sect./subsect. shared some common molecular and cytogenetic features. The results indicated that sect. Martagon is monophyletic in a single clade; Lophophorum should be treated as a different taxon from Sinomartagon; Lilium taliense and Lilium wardii Stapf ex Stern show close molecular and cytological phylogenetic relationships; Lilium duchartrei Franch. is more distantly related to other sect. Sinomartagon species; Lilium lophophorum (Bureau & Franch.) Franch. is distinct from other Lophophorum species; Leucolirion species form two relatively independent groups (6a and 6b, with Lilium henryi Baker and Lilium rosthornii Diels in 6a); and Nomocharis-like Lilium species should be treated as a different taxon, like sect. Lophophorum. Based on previous studies and our findings, we discuss the evolutionary trends of Lilium with references to its geographical distribution. We propose that sect. Lophophorum is a relatively primitive group, and species in subsect. Leucolirion 6b may be more evolved than those in 6a. These findings provide a new reference for the taxonomy, phylogeny, and breeding of Lilium.

Grey mould is one of the most determinative factors of lily growth and plays a major role in limiting lily productivity. MicroRNA159 (miR159) is a highly conserved microRNA in plants, and participates in the regulation of plant development and stress responses. Our previous studies revealed that lre‐miR159a participates in the response of Lilium regale to Botrytis elliptica according to deep sequencing analyses; however, the response mechanism remains unknown. Here, lre‐miR159a and its target LrGAMYB gene were isolated from L. regale. Transgenic Arabidopsis overexpressing lre‐MIR159a exhibited larger leaves and smaller necrotic spots on inoculation with Botrytis than those of wild‐type and overexpressing LrGAMYB plants. The lre‐MIR159a overexpression also led to repressed expression of two targets of miR159, AtMYB33 and AtMYB65, and enhanced accumulation of hormone‐related genes, including AtPR1, AtPR2, AtNPR1, AtPDF1.2, and AtLOX for both the jasmonic acid and salicylic acid pathways. Moreover, lower levels of H2O2 and O2- were observed in lre‐MIR159a transgenic Arabidopsis, which reduced the damage from reactive oxygen species accumulation. Taken together, these results indicate that lre‐miR159a positively regulates resistance to grey mould by repressing the expression of its target LrGAMYB gene and activating a defence response., lre‐miR159a positively regulates resistance to grey mould by repressing the expression of its target LrGAMYB gene and activating a defence response in Lilium regale.

Polyploids generally show strong environmental adaptation and diverse morphological variations. Therefore, polyploid induction is an important protocol for plant breeding. Lilium rosthornii is a wild lily species with high horticultural value and excellent disease resistance. In this study, seeds of L. rosthornii were subjected to polyploidy induction treatments to obtain multiple genotypes of tetraploids. Germinated seeds were immersed in two antimitotic agents at different concentrations for various times. In total, 199 tetraploid genotypes were obtained. The most efficient treatments of each agent were immersed in 0.05% colchicine for 36 h and in 0.01% oryzalin for 24 h; the induction rate of the former (27.78%) was significantly higher than that of the latter (22.22%). The swollen hypocotyl phenotype after colchicine and oryzalin treatments was strongly correlated with tetraploidy (0.989** and 0.975**, respectively), suggesting that this phenotype could serve as an early ploidy selection trait. The correlations were weaker between stomata length/density and tetraploidy (0.773** and 0.695**, respectively), implying that stomatal characters are affected by both the ploidy level and genotype. After several rounds subculture in vitro, the morphology and growth traits were not significantly different between diploids and tetraploids, but there were wider variations in these parameters in tetraploids than in diploids. After transplanting, the bulblet germination rate was higher in tetraploids than in diploids. The leaf phenotype did not differ between tetraploids and diploids initially, but the leaves of tetraploids became larger than those of diploids over time. Together, these suggest that tetraploids, may contribute diverse characteristics to lily breeding. Multiple genotypes of tetraploid Lilium rosthornii were induced by colchicine and oryzalin. Swollen hypocotyl was associated with polyploidy. Compared with diploids, tetraploids showed wider variations in morphological parameters.

Lilium × formolongi is an important cut flower species that is able to flower within a year following seed propagation, with flower induction that is very sensitive to the photoperiod. Cryptochromes are blue/UV-A light receptors that regulate many important plant growth and development processes, including photoperiodic flowering. In this study, we isolated the cryptochrome 1 (CRY1) gene from L. × formolongi and analyzed its function in transgenic Arabidopsis. The predicted LfCRY1 protein was strongly homologous to other CRY1 proteins. The transcription of LfCRY1 was induced by blue light, with LfCRY1 exhibiting its highest expression and diurnal expression patterns during the flowering-induction stage under both long-day (LD) and short-day (SD) photoperiods. Overexpression of LfCRY1 in Arabidopsis promoted flowering under LDs but not SDs and inhibited hypocotyl elongation under blue light. The LfCRY1 protein was located in both the nucleus and cytoplasm. LfCRY1 interacted with the important flowering activator LfCOL9 in both yeast and onion cells. These results provide functional evidence for the role of LfCRY1 in controlling photoperiodic flowering under LDs and indicate that LfCRY1 may be a counterpart of AtCRY1. Understanding the role of LfCRY1 in photoperiodic flowering is beneficial for the molecular breeding of lilies with shorter vegetative stages.

Crossing between Lilium OT (Oriental × Trumpet) cultivars and L. regale could provide an accessible way to develop lily cultivars with both horticultural traits and stress resistance. Physical mapping of 45S ribosomal DNA (rDNA) loci by fluorescence in situ hybridization (FISH) can help understand genetic relationships and chromosome inheritance in lily breeding. In this study, hybrid progeny were obtained from the combinations of ‘Conca d’Or’ × L. regale and ‘Saltarello’ × L. regale, confirming the fertility of OT cultivars as female parents in crosses with L. regale. ‘Saltarello’ × L. regale had the highest embryo rate (8.96), indicating cross-compatibility between ‘Saltarello’ and L. regale. Based on the results of crossing and FISH, the genetic relationship between OT cultivars and L. regale was further investigated. ‘Saltarello’ possessed twelve 45S rDNA signal loci, of which eight were similar to those of L. regale, on chromosomes 1, 2 and 5, confirming the closest genetic relationship with L. regale, ‘Conca d’Or’ with five similar sigals second and ‘Manissa’ with three similar sigals farthest. The 45S rDNA loci in hybrids of ‘Saltarello’ ×L. regale were located on chromosomes 1 through 6 and varied in number from 7 to 13. These hybrids included both euploids and aneuploids, demonstrating that allotriploid Lilium OT hybrids can produce viable gametes with euploid and aneuploid chromosome numbers. Thus, it should be possible to select new cultivars from interspecific hybrids of OT hybrids × L. regale.

Rosa chinensis Jacq., originating from China, occupied a pivotal position in the floral industry all over the world. Drought and soil salinization is a major problem in the Chinese rose planting industry. Therefore, the study on resistance is important to improve R. chinensis breeding. In this work, leaves of twelve samples with three biological repetitions were collected for transcriptome sequencing. In total 83.14 Gigabyte (G) Illumina sequence data were generated and 74 850 unigenes were successfully annotated. Differentially expressed gene (DEG) analysis was performed by comparing the transcriptomes of the 0, 2, 4 and 7 days salt treatment, based upon which several potential candidate genes involved in salt tolerance response had been identified. DEGs involved in plant hormone signal transduction, photosynthesis, and catalytic activity were examined in depth. The experimental result indicated that the R. chinensis showed strong stress signals after being subjected to salt stress. In the initial stage of salt treatment (two days), plant protective process began to take effect, which was embodied in the regulation of oxidoreductase activities and plant hormone signal transduction pathway. In addition, the expression level of P5CS was up-regulated in R. chinensis, but the proline metabolic pathway was not affected. In the late stage of salt treatment (four and seven days), the photosynthesis – antenna proteins pathway has been greatly affected, and the down-regulated PYL, Snrk2 and MRP lead to the salt tolerance decline, which was consistent with the leaf wilting performance of the R. chinensis in four and seven days of treatment. All the candidate genes were verified by RT-qPCR.

Background MicroRNAs play pivotal roles in plant vegetative phase change and flowering induction via integrating into multiple flowering pathways. Lilium × formolongi is an important ornamental lily cultivar that can flower within one year after sowing. However, it remains unresolved how miRNA-mediated regulation networks contribute to the L. × formolongi characteristics of a short vegetative growth period and rapid flowering. Results In this study, the small RNA libraries and one degradome library were constructed for L. × formolongi during vegetative growth and flowering initiation, and 366 conserved miRNAs and 32 novel miRNAs were identified. Additionally, 84 miRNAs were significantly differentially expressed during development. A total of 396 targets of 185 miRNAs were identified and validated through degradome sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that functions of the targets were top enriched in the cold and cadmium ion responses, pentose phosphate pathway and carbon fixation in photosynthetic organisms. Furthermore, among 23 differentially expressed miRNA-target pairs, the miR156s-LfSPL2, miR172a-LfAP2 and miR164a-LfNAC pairs as well as miR159a-LfSPL2 were found to be relevant to flowering based on the correlation analysis of expression profiles in the miRNA libraries, degradome and transcriptome. A coexpression regulatory network focused on differentially expressed pairs was also constructed by WGCNA, and 14 miRNAs were considered putative key miRNAs during vegetative development and flowering induction. miR156a/ d/ e showed particularly strong relationships with other miRNAs in the coexpression network. Conclusions This study provides cues for the further exploration of the regulatory mechanisms of short vegetative development and flowering in L. × formolongi.

Lilium × formolongi, a facultative long-day (LD) plant, can complete the floral transition within one year after sowing under LD conditions. In addition to the photoperiod, the molecular mechanisms by which other flowering regulators, such as sugar, participate in juvenile development and flowering induction in L. × formolongi remain elusive. Therefore, based on the investigation of seedling development under different day length conditions, we explored the growth and nonstructural carbohydrate (NSC) contents of leaves and underground bulbs. Furthermore, the expression profiles of trehalose-6-phosphate synthase (TPS)-coding genes, LfTPSs, and miR156 were also determined. Three putative LfTPS genes, LfTPS1, LfTPS3 and LfTPS5, displayed high expression levels at the juvenile vegetative stage under different day length conditions. Among them, LfTPS1 maintained gradually elevated expression until the visible bud stage under short-day (SD) conditions. Additionally, the expression levels of LfTPS3 and LfTPS5 increased with the exogenous sucrose concentration. In contrast, the expression of miR156 rapidly decreased under the same sucrose treatments. Overexpression of LfTPS1/3/5 hastened flowering and the decline in miR156 expression levels to varying degrees in transgenic Arabidopsis. Taken together, the results demonstrate that LfTPS1, LfTPS3 and LfTPS5 modulate both juvenile vegetative development and flowering induction controlled by sugars in L. × formolongi.

Anthocyanins are one of the main components of pigments, that are responsible for a wide range of colors in plants. To clarify the regulatory mechanism of anthocyanin biosynthesis in oriental hybrid lily, UPLC/MS analysis was performed to identify the pigments in two cultivars (white and pink). Four major anthocyanins were identified in pink cultivar, and no anthocyanins were detected in white cultivar. Transcriptome and small RNA sequencing (sRNAseq) analyses were performed using tepal tissues at two floral developmental stages from the two cultivars. In total, 55,698 transcripts were assembled, among which 233 were annotated as putative anthocyanin-related transcripts. Differential expression analysis and qRT-PCR results confirmed that most of the anthocyanin-related structural genes had higher expression levels in pink cultivar than in white cultivar. Conversely, LhANR showed a significantly high expression level in white cultivar. Annotated transcription factors (TFs), including MYB activators, MYB repressors and bHLHs, that putatively inhibit or enhance the expression of anthocyanin-related genes were identified. LhMYBA1, an anthocyanin activator, was isolated, and its heterologous expression resulted in a remarkably high level of anthocyanin accumulation. Additionally, 73 differentially expressed microRNAs (miRNAs), including 23 known miRNAs, were detected through sRNAseq. The miRNA target prediction showed that several anthocyanin-related genes might be targeted by miRNAs. Expression profile analysis revealed that these miRNAs showed higher expression levels at later floral developmental stages in white cultivar than in pink cultivar. The results indicated that anthocyanin deficiency in white cultivar might be influenced by multiple levels of suppressive mechanisms, including mRNAs and sRNAs.

Lilium is an important commercial flowering species, and there are many varieties and more than 100 species of wild Lilium. Lilium × formolongi is usually propagated from seedlings, and the flowering of these plants is driven mainly by the photoperiodic pathway. Most of the other lily plants are propagated via bulblets and need to be vernalized; these plants can be simply divided into pretransplantation types and posttransplantation types according to the time at which the floral transition occurs. We identified three Lilium FLOWERING LOCUS T (LFT) family members in 7 Lilium varieties, and for each gene, the coding sequence of the different varieties was identical. Among these genes, the LFT1 gene of Lilium was most homologous to the AtFT gene, which promotes flowering in Arabidopsis. We analyzed the expression patterns of LFT genes in Lilium × formolongi seedlings and in different Lilium varieties, and the results showed that LFT1 and LFT3 may promote floral induction. Compared with LFT3, LFT1 may have a greater effect on floral induction in Lilium, which is photoperiod sensitive, while LFT3 may play a more important role in the floral transition of lily plants, which have a high requirement for vernalization. LFT2 may be involved in the differentiation of bulblets, which was verified by tissue culture experiments, and LFT1 may have other functions involved in promoting bulblet growth. The functions of LFT genes were verified by the use of transgenic Arabidopsis thaliana plants, which showed that both the LFT1 and LFT3 genes can promote early flowering in Arabidopsis. Compared with LFT3, LFT1 promoted flowering more obviously, and thus, this gene could be an important promoter of floral induction in Lilium.

A major production constraint of lilies is gray mold caused by the necrotrophic fungus Botrytis elliptica. The molecular basis of lily plant resistance to B. elliptica remains largely unexplored. To systematically dissect transcriptomic responses, we constructed four RNA-seq libraries from the leaves of Lilium regale, a promising Chinese wild Lilium species, after B. elliptica infection for 0, 4, 12, or 24 h. The sequence reads were assembled into 96 416 unigenes, of which 7697 were differentially expressed. Profiling analysis revealed changes in gene expression, including 2261 downregulated genes and 3599 upregulated genes. Quantitative real-time polymerase chain reaction analysis of 39 defense-related unigenes confirmed that the transcriptional changes of these genes presented in the RNA-seq data were predominately affected by B. elliptica infection. Key B. elliptica modulated genes played roles in defense responses mediated by phytohormones involved in jasmonate signaling, whereas salicylic acid and ethylene were not involved. Among transcription factors, some WRKYs, MYB, and ethylene responsive factor were clearly upregulated. Additionally, a group of genes encoding known important defense-related proteins, such as receptor-like kinases, antioxidant enzymes, polyphenol oxidase, pathogenesis-related proteins, and proteins associated with phenylpropanoid metabolism, exhibited high transcript abundances. Furthermore, the expressions of selected candidate genes were induced more rapidly and strongly in the resistant genotype than in the susceptible genotype. Taken together, the results provide a better understanding of the defense responses involved in the crosstalk between the lily and B. elliptica and a valuable set of sequence data for gray mold resistant candidate gene discovery.

Lilium�formolongi is a facultative long-day (LD) plant. Aiming to dissect the molecular regulation of the photoperiodic pathway, largely unknown in Lilium, we explored the CONSTANS/FLOWERING LOCUS T (CO/FT) module, a major regulatory factor in the external coincidence model of the photoperiodic flowering pathway in lily. We identified eight CONSTANS-LIKE (COL) family members in L.�formolongi, which could be divided into three types, according to their zinc-finger (B-box) protein domains. Type I included only LfCOL5, containing two B-box motifs. Type II contained six LfCOLs members that had only one B-box motif. Type III contained only LfCOL9 that showed a normal B-box and a second divergent B-box motif. Phylogenic analyses revealed that LfCOL5 was the closest to Arabidopsis CO. LfCOL5, LfCOL6 and LfCOL9 were up-regulated at the flowering induction stage under LDs, coinciding with an increase in LfFT1 expression. LfCOL5, LfCOL6 and LfCOL9 also showed obvious diurnal expression pattern for 3 d under LDs. However, under short-day (SD) conditions, the expression patterns of LfCOL5, LfCOL6 and LfCOL9 were variable and complex, with regard to the developmental stages and circadian rhythm. LfCOL5, LfCOL6 and LfCOL9 complemented the late flowering phenotype of the co mutant in Arabidopsis. Taken together, the results suggest that LfCOL5, LfCOL6 and LfCOL9 are involved in triggering flowering induction under LDs. LfCOL6 and LfCOL9 belong to types different from functional COL homologs in other plant species, illustrating the variation in phylogeny, evolution and gene function among LfCOL family members.

A major constraint in producing lilies is gray mold caused by Botrytis elliptica and B. cinerea. WRKY transcription factors play important roles in plant immune responses. However, limited information is available about the WRKY gene family in lily plants. In this study, 23 LrWRKY genes with complete WRKY domains were identified from the Botrytis-resistant species Lilium regale. The putative WRKY genes were divided into seven subgroups (Group I, IIa-e, and III) according to their structural features. Sequence alignment revealed that LrWRKY proteins have a highly conserved WRKYGQK domain and a variant, the WRKYGKK domain, and these proteins generally contained similar motif compositions throughout the same subgroup. Functional annotation predicted they might be involved in biological processes related to abiotic and biotic stresses. A qRT-PCR analysis confirmed that expression of six LrWRKY genes in L. regale or the susceptible Asian hybrid 'Yale' was induced by B. cinerea infection. Among these genes, LrWRKY4, LrWRKY8 and LrWRKY10 were expressed at a higher level in L. regale than 'Yale', while the expression of LrWRKY6 and LrWRKY12 was lower in L. regale. Furthermore, LrWRKY4 and LrWRKY12 genes, which also respond to salicylic acid (SA) and methyl jasmonate (MeJA) treatments, were isolated from L. regale. Subcellular localization analysis determined that they were targeted to the nucleus. Constitutive expression of LrWRKY4 and LrWRKY12 in Arabidopsis resulted in plants that were more resistant to B. cinerea than wild-type plants. This resistance was coupled with the transcriptional changes of SA and JA-responsive genes. Overall, our study provides valuable information about the structural and functional characterization of LrWRKY genes that will not only deepen our understanding of the molecular mechanisms underlying the defense of lily against B. cinerea but also offer potential targets for cultivar improvement via biotechnology.

Lilium × formolongi, a facultative long-day (LD) plant, has precocious flowering characteristics, as it can flower within one year from seeds without the need for vernalization. Under normal LD conditions, the seedlings entered the flowering induction phase and bolted with 9−10 rosette leaves at 6 months after sowing. High temperature, especially at the root zone, inhibited bolting of the seedlings in LDs, accompanied by changes in plant shape and rapid expansion of underground bulbs. Low temperature in the natural environment could rapidly reverse the inhibition caused by high temperature, and promote bolting even under non-induced short days. The circadian clock and photoperiod pathways were disturbed at the transcriptional level by high temperature in both leaves and bulbs, such as LfCCA1, LfCRY, LfCOP1, LfFKF1, LfCOLs and LfPIFs. Thus, the flowering integrators LfFT1 and LfSOC1 were suppressed by high temperature. To determine the flowering inhibitors induced by high temperature in the root zone, the metabolome was analyzed under different conditions. ABA and ACC accumulation in the leaves and bulbs respectively during high temperature, was crucial to flowering inhibition possibly via interaction with the circadian clock and photoperiod-dependent pathways. Additionally, under high temperature conditions, D-allose, pentose and glucuronate interconversions and glutathione metabolism in the leaves were significantly activated. After cold exposure, TCA cycle was suppressed and SA, JA and some sugar (D-mannose, D-fructose, D-arabinose and maltotriose) levels were upregulated in the leaves, simultaneously with the vigorous fatty acid metabolism in the bulbs. These metabolic regulations were crucial to the phase transition under different conditions.

The photoperiodic flowering pathway is essential for plant reproduction. As blue and ultraviolet-A light receptors, cryptochromes play an important role in the photoperiodic regulation of flowering. Lilium × formolongi is an important cut flower that flowers within a year after seed propagation. Floral induction is highly sensitive to photoperiod. In this study, we isolated the CRYPTOCHROME2 gene (LfCRY2) from L. × formolongi. The predicted LfCRY2 protein was highly homologous to other CRY2 proteins. The transcription of LfCRY2 was induced by blue light. LfCRY2 exhibits its highest diurnal expression during the floral induction stage under both long-day and short-day photoperiods. Overexpression of LfCRY2 in Arabidopsis thaliana promoted flowering under long days but not short days, and inhibited hypocotyl elongation under blue light. Furthermore, LfCRY2 was located in the nucleus and could interact with L. × formolongi CONSTANS-like 9 (LfCOL9) and A. thaliana CRY-interacting basic-helix-loop-helix 1 (AtCIB1) in both yeast and onion cells, which supports the hypothesis that LfCRY2 hastens the floral transition via the CIB1-CO pathway in a manner similar to AtCRY2. These results provide evidence that LfCRY2 plays a vital role in promoting flowering under long days in L. × formolongi.

Photosynthesis play an important role in plant growth. Generally, it will be increased with increased DNA content per cell. However, in organ levels, the changes of photosynthesis are species dependent. So, for target breeding materials, chromosome duplication how to influence photosynthetic performances need further investigation. In this study, three lily (Lilium) species, L. rosthornii (LRI), L. primulinum var. orchraceun (LPO) and L. regale (LRE), and their colchicine-induced autotetraploids were employed in comparing photosynthetic related characteristics. For anatomic traits, the number of palisade mesophyll cells was significantly increased in LRI and LPO but decreased in LRE after polyploidization. Moreover, except the intercellular space decreased, the mesophyll cell sizes and shapes presented different changes in the three species after polyploidization. For physical-biochemical traits, the chloroplast number, thylakoid grana thickness, and photosynthetic pigments and efficiency were significantly increased in LRI after polyploidization. For LPO, the chloroplast number, and weak light absorbed and CO2 assimilated rates were significantly increased, while others showed no significant changes after polyploidization. Out of expecting, for LRE, the chloroplast number and photosynthetic pigments were significantly decreased after polyploidization. Moreover, the structures of chloroplasts were abnormal with extremely thicker thylakoid grana and more tightly arranged thylakoid layer in tetraploids than in diploids. The photosynthetic rates of LRE were similar in diploid and tetraploid in natural conditions, while the maximum photosynthetic rate (Pmax) of light-/CO2-response curves were higher in diploids than in diploids. After plants withered, larger bulblet with more developed root systems were well observed from the three tetraploid species.

The onset of flowering is critical for the reproductive development of plants. Lilium × formolongi is a lily hybrid that flowers within a year after sowing. We successfully identified four important stages during vegetative growth and flowering initiation of L. × formolongi under long day conditions. The plant tissues from the four stages were used in a genome-wide transcriptional analysis to investigate stage-specific changes of gene expression in L. × formolongi. In total, the sequence reads of the four RNA-sequencing libraries were assembled into 52,824 unigenes, of which 37,031 (70.10%) were differentially expressed. The global expression dynamics of the differentially expressed genes were predominant in flowering induction phase I and the floral differentiation stage, but down-regulated in flowering induction phase II. Various transcription factor families relevant to flowering were elucidated, and the members of the MADS-box, SBP and CO-like transcription factor families were the most represented. There were 85 differentially expressed genes relevant to flowering. CONSTANS-LIKE, FLOWERING LOCUS T, TREHALOSE-6-PHOSPHATE SYNTHASE and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE homologs were discovered and may play significant roles in the flowering induction and transition process of L. × formolongi. A putative gene regulatory network, including photoperiod, age-dependent and trehalose-6-phosphate flowering pathways, was constructed. This is the first expression dataset obtained from a transcriptome analysis of photoperiod-mediated flowering pathway in lily, and it is valuable for the exploration of the molecular mechanisms of flowering initiation and the short vegetative stage of L. × formolongi.

Lilium martagon var. pilosiusculum is an endangered species with high ornamental value in Xinjiang province (China). In this study, we reported a complete chloroplast genome of L. martagon var. pilosiusculum, which was de novo assembled using the next-generation sequencing data. The complete chloroplast genome is 152,816 in length, including a large single copy region of 82,265 bp and a small single copy region of 17,541 bp and two inverted repeat regions of 26,505 bp. A total of 110 functional genes were encoded, consisting of 76 protein-coding genes, 30 transfer RNA genes, and four ribosomal RNA genes. The overall AT content of the chloroplast genome is 63.00%. In addition, 68 SSRs and 31 large repeat sequences were found. In the maximum likelihood tree, a strong phylogenetic signal showed that L. martagon var. pilosiusculum is a species of lilium.

Proline accumulations in abiotically stressed plants is generally considered to benefit their stress tolerance. The Δ1-Pyrroline-5-carboxylate synthetase (P5CS) gene family, which encodes the rate-limiting enzyme in proline biosynthesis pathway, usually contains two duplicated genes in most plants. However, three P5CS genes including LrP5CS1, LrP5CS2 as well as a third one, LrP5CS3, were isolated from Lilium regale. LrP5CS3 is highly identical to LrP5CS1 in amino acid sequences, indicating they could come from a paralogous duplication. The phylogenetic tree suggested that the duplication of LrP5CS occurred independently after the divergence of Liliales and commelinoids. The expression of LrP5CS1 was strongly induced in leaves and roots both under drought and salinity, while that of LrP5CS3 was upregulated more moderately. LrP5CS2 stayed almost constitutive under stress. LrP5CS1 exhibited the highest activity after expressed in E. coli. Overexpression of LrP5CS genes conferred enhanced osmotic, drought and salt tolerance on transgenic Arabidopsis without negative effects in unstressed condition. Under salt stress, lines LrP5CS2 accumulated fewer proline than others, and lines LrP5CS1 grew better in root elongation. The roots of lines LrP5CS3 grew better than all others under unstressed condition and osmotic stress. Our study suggests that the three LrP5CS genes play distinct roles respectively in proline accumulation and abiotic stress tolerance.

Polyploidy plays an important role in plant breeding, inducing a wide range of effects on photosynthetic performance, cellular and photochemical aspects of photosynthesis. Although the impacts of polyploidization on photosynthesis have been examined in several genera, comparatively little is known about the photosynthetic responses to polyploidy in Lilium. In this study, diploid and colchicine-induced tetraploid Lilium FO hybrids were used to study the effects of polyploidization on cellular, photochemical and photosynthetic characteristics in Lilium. Polyploidization caused a significant change in leaf anatomical structure, chloroplast ultrastructure and photosynthetic pigment contents. Thicker epidermal and spongy tissue, more and thicker thylakoid lamellae and higher chlorophyll and carotenoid contents were observed in the tetraploid than in the diploid. More regularly and tightly arranged thylakoids were closely associated with more efficient light-harvesting machinery. And the tetraploid plants showed a higher net photosynthetic rate (Pn) and maximum net photosynthetic rate (Pmax) than the diploid plants, under both natural conditions and gradients of light intensity/CO2 concentration. In addition, the tetraploid had a significantly higher light saturation point (LSP), but a lower light compensation point (LCP) than the diploid, indicating that tetraploid plants acquired light energy more efficiently. These results suggested that cellular and photochemical alterations caused by polyploidization improve the capacity for light absorption and conversion and further enhanced the photosynthetic performance in Lilium FO hybrids.

Plants regulate responses to salt stress using biological pathways, such as signal perception and transduction, photosynthesis, and energy metabolism. Little is known about the genetics of salt tolerance in Rosa chinensis. Tineke and Hiogi are salt-tolerant and salt-sensitive varieties of R. chinensis, respectively, and are good choices for studying salt-tolerance genes. We studied leaf and root tissues from 1-year-old Hiogi and Tineke plants simultaneously grown under the same conditions. A 0.4%-mmol/L salt ion mixture was added to the basic growth medium. Illumina sequencing was used to identify differentially expressed transcripts. GO and KEGG pathway enrichment analyses were performed to identify differentially expressed genes. We identified many differentially expressed genes associated with salt tolerance. The abscisic acid-dependent signaling pathway was the main pathway that mediated the salt stress response in R. chinensis. Two pathways (plant hormone signal transduction and glutathione metabolism) were also active in salt stress responses in R. chinensis. The difference in salt tolerance in the cultivars was due to different gene sensitivity to salt in these two pathways. Roots also play a role in salt stress response. The effects of salt stress in the roots are eventually manifested in the leaves, causing changes in processes such as photosynthesis, which eventually result in leaf wilting. In Tineke, Snrk2, ABF, HSP, GSTs, and GSH1 showed high activity during salt stress, indicating that these genes are markers of salt tolerance.

Gray mold disease, caused by the fungus Botrytis elliptica, is one of the major diseases affecting Lilium species, and it has become a limiting factor in the production of ornamental lilies. To support selecting and breeding Botrytis-resistant cultivars, a total of 50 Lilium cultivars belonging to seven hybrid types were evaluated using a detached leaf technique for resistance to B. elliptica. Through resistance evaluations, Oriental × Trumpet and Oriental hybrid cultivars were classified as resistant lines, while Asiatic and Trumpet hybrids were classified as susceptible lines. A highly resistant (HR) Oriental hybrid, ‘Sorbonne’, and a highly susceptible (HS) Asiatic hybrid, ‘Tresor’, were selected for further study of early host-parasite interactions. After infection, B. elliptica grew faster and more easily on the leaves of ‘Tresor’ than on those of ‘Sorbonne’ during initial infection; when ‘Tresor’ leaves were completely infected, only a few lesions were observed on ‘Sorbonne’ leaves. Biochemical differences between these two cultivars were found following inoculation with B. elliptica, as shown by studies of reactive oxygen species (ROS) and the enzymatic antioxidant system. Rapid accumulation of H2O2 and ·O2− to trigger a defense response was detected in HR ‘Sorbonne’. Meanwhile, higher levels of antioxidant activity, including SOD, POD and CAT, were found in HR ‘Sorbonne’ than in HS ‘Tresor’ before 48 h post-inoculation, but antioxidant activity was reduced with subsequent infection progress. These large and timely increases in ROS and antioxidant activities could be the main contributors to the high resistance of the ‘Sorbonne’ cultivar.

Plant anthocyanin biosynthesis is regulated by a combination of transcription factors. Here, exogenous anthocyanin regulator genes Delila/Rosea1 (Del/Ros1) or Bperu/Colored aleurone1 (B1/C1) were i...

Lily is an important cut-flower and bulb crop in the commercial market. Here, transcriptome profiling of Lilium 'Sorbonne' was conducted through de novo sequencing based on Illumina platform. This research aims at revealing basic information and data that can be used for applied purposes especially the molecular regulatory information on flower color formation in lily. In total, 36,920,680 short reads which corresponded to 3.32 GB of total nucleotides, were produced through transcriptome sequencing. These reads were assembled into 39,636 Unigenes, of which 30,986 were annotated in Nr, Nt, Swiss-Prot, KEGG, COG, GO databases. Based on the three public protein databases, a total of 32,601 coding sequences were obtained. Meanwhile, 19,242 Unigenes were assigned to 128 KEGG pathways. Those with the greatest representation by unique sequences were for ''metabolic pathways'' (5,406 counts, 28.09 %). Our transcriptome revealed 156 Unigenes that encode key enzymes in the flavonoid biosynthesis pathway including CHS, CHI, F3H, FLS, DFR, etc. MISA software identified 2,762 simple sequence repeats, from which 1,975 primers pairs were designed. Over 2,762 motifs were identified, of which the most frequent was AG/CT (659, 23.86 %), followed by A/T (615, 22.27 %) and CCG/CGG (416, 15.06 %). Based on the results, we believe that the color formation of the Lilium 'Sorbonne' flower was mainly controlled by the flavonoid biosynthesis pathway. Additionally, this research provides initial genetic resources that will be valuable to the lily community for other molecular biology research, and the SSRs will facilitate marker-assisted selection in lily breeding.

MicroRNAs, as master regulators of gene expression, have been widely identified and play crucial roles in plant-pathogen interactions. A fatal pathogen, Botrytis elliptica, causes the serious folia disease of lily, which reduces production because of the high susceptibility of most cultivated species. However, the miRNAs related to Botrytis infection of lily, and the miRNA-mediated gene regulatory networks providing resistance to B. elliptica in lily remain largely unexplored. To systematically dissect B. elliptica-responsive miRNAs and their target genes, three small RNA libraries were constructed from the leaves of Lilium regale, a promising Chinese wild Lilium species, which had been subjected to mock B. elliptica treatment or B. elliptica infection for 6 and 24 h. By high-throughput sequencing, 71 known miRNAs belonging to 47 conserved families and 24 novel miRNA were identified, of which 18 miRNAs were downreguleted and 13 were upregulated in response to B. elliptica. Moreover, based on the lily mRNA transcriptome, 22 targets for 9 known and 1 novel miRNAs were identified by the degradome sequencing approach. Most target genes for elliptica-responsive miRNAs were involved in metabolic processes, few encoding different transcription factors, including ELONGATION FACTOR 1 ALPHA (EF1a) and TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR 2 (TCP2). Furthermore, the expression patterns of a set of elliptica-responsive miRNAs and their targets were validated by quantitative real-time PCR. This study represents the first transcriptome-based analysis of miRNAs responsive to B. elliptica and their targets in lily. The results reveal the possible regulatory roles of miRNAs and their targets in B. elliptica interaction, which will extend our understanding of the mechanisms of this disease in lily.

China is known as an important distribution center for the genus Lilium, with 55 species having been reported in the country thus far. This study was conducted to provide the first comprehensive evaluation of Lilium distribution in China. A total of 27 species of Lilium and 3 varieties were found to exist in the main centers of Lilium distribution in China; these species were identified and classified based on their morphological characteristics. The distribution and botanical characteristics of the various species are reviewed in this study. We also describe differences, mainly in flower color within the same species, which may be representative of new natural varieties or ecotypes. An analytical hierarchy process (AHP) utilizing 16 indicator characteristics was used to evaluate the collected Lilium resources based on their ornamental value, utilization potential, and ecological adaptability. L. dauricum received the highest AHP scores, indicating that it was the species best suited for sustained exploitation. In addition, L. henryi, L. rosthornii, L. tsingtauense, L. concolor var. pulchellum, L. lancifolium, and species with trumpet–shaped flowers also showed better potential for exploitation than other species. This study also provides references regarding the effective exploitation and use of wild Lilium resources.

Normalization to reference genes is the most common method to avoid bias in real-time quantitative PCR (qPCR), which has been widely used for quantification of gene expression. Despite several studies on gene expression,Lilium, and particularlyL. regale, has not been fully investigated regarding the evaluation of reference genes suitable for normalization. In this study, nine putative reference genes, namely18S rRNA,ACT,BHLH,CLA,CYP,EF1,GAPDH,SANDandTIP41, were analyzed for accurate quantitative PCR normalization at different developmental stages and under different stress conditions, including biotic (Botrytis elliptica), drought, salinity, cold and heat stress. All these genes showed a wide variation in their Cq (quantification Cycle) values, and their stabilities were calculated by geNorm, NormFinder and BestKeeper. In a combination of the results from the three algorithms,BHLHwas superior to the other candidates when all the experimental treatments were analyzed together;CLAandEF1were also recommended by two of the three algorithms. As for specific conditions,EF1under various developmental stages,SANDunder biotic stress,CYP/GAPDHunder drought stress, andTIP41under salinity stress were generally considered suitable. All the algorithms agreed on the stability ofSANDandGAPDHunder cold stress, while onlyCYPwas selected under heat stress by all of them. Additionally, the selection of optimal reference genes under biotic stress was further verified by analyzing the expression level ofLrLOXin leaves inoculated withB. elliptica. Our study would be beneficial for future studies on gene expression and molecular breeding ofLilium.

Protocols for in vitro regeneration and production of in vitro-propagated plants and a transformation system were developed for Mirabilis jalapa (Nyctaginaceae). Among the types of explants and the different media tested, consistent shoot regeneration was obtained only from nodal segments grown in a regeneration medium consisting of Murshashige and Skoog medium supplemented with 2 mg l−1 6-benzyladenine, 2 mg l−1 zeatin and 1 mg l−1 indole acetic acid. Regeneration efficiency was dependent on the type of plant – white or pink flowers – used as the source of explants. Stable transformation was obtained following inoculation of nodal segments with Agrobacterium tumefasciens strain EHA105, which harbours the binary plasmid pAD1339 containing both nptII and gus genes under the control of the 35S promoter. Transformation was confirmed by PCR and Southern blot analysis of genomic DNA from mature regenerated plants. β-Glucuronidase (GUS) activity was observed only in tissues regenerated from in vitro-grown plants and not in tissues originating from greenhouse-grown plants. GUS expression was not uniform in regenerated leaves and showed a chimera pattern.

Recent molecular and karyologic studies have significantly modified delimitation of Lilium. However, despite the importance of pollen evolution in the genus comprehensive studies with electron microscopy and evaluation of pollen evolution are lacking. Therefore, we studied pollen morphology in a sample of 65 individuals from 37 taxa covering all the sections distributed in the world, using scanning electron microscopy. Our collection of 49 individuals from 21 taxa covering all five sections in China was also included in the database. We found pollen tetrads in L. bakerianum. Based on present and previous studies, our results suggest that pollen from L. formosanum should be classified as a new type, Formosanum. Combined with morphological and molecular evidence, pollen sculpture patterns appear to reflect phylogenetic relationships and are useful for species or subsection delimitation. Based on a comprehensive survey and correlation with potential functional implications, we propose the following hypothesis: evolution of an exine sculpture shows pollen type trends from Martagon → Callose → Concolor → Formosanum. The evolutionary trend regarding pollen sculpture and size could be related to selective pressure to adapt to environmental conditions. Pollen size and shape showed a significantly positive correlation with annual precipitation, and smaller pollen grains appear to adapt better in habitats with extreme conditions. Evolution trends in exine sculpture do not appear to be definitively correlated with pollen size and shape.

We present a comprehensive phylogeny derived from nuclear ribosomal DNA (nrDNA) for 214 samples representing 98 species and five varieties, including 44 species and five varieties native to China. Our collection of 25 species and five varieties (44 samples) covering all five sections of the genus (Comber) distributed in China also were included in the internal transcribed spacer (ITS) database. This study incorporates previous research with an emphasis on Chinese species, including the controversial subsection, Sinomartagon 5c Comber. In the phylogenetic tree obtained by maximum parsimony (PAUP) and maximum likelihood (RAxML) analyses, the samples were divided into four major groups. Our results suggest that the subsection (subsect.) 5c Comber should be classified into the true subsect. 5c and the section (sect.) Lophophorum. And the latter was divided into three subsections (subsect. Lophophorum I, subsect. Lophophorum II, and subsect. Lophophorum III). Based on molecular phylogenetic analysis and fluorescence in situ hybridization, we report that L. henryi and L. rosthornii are closely related, and we propose their classification into subsect. Leucolirion 6a. Our results support Comber's subdivision of sect. Leucolirion, which was primarily based on bulb color. Chinese species were divided into five sections: sect. Martagon, sect. Archelirion, sect. Leucolirion, sect. Sinomartagon, and sect. Lophophorum. These findings contribute to our understanding of the phylogeny, origin, and classification of Lilium.