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.

Integrated mRNA and small RNA sequencing reveals a regulatory network associated with flower color in oriental hybrid lily.

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.

Functional analysis and expression patterns of members of the FLOWERING LOCUS T (FT) gene family in Lilium.

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.

Genome-Wide Analysis of the Cyclin Gene Family and Their Expression Profile in Medicago truncatula.

Cyclins, together with highly conserved cyclin-dependent kinases (CDKs), play an important role in the process of cell cycle in plants, but less is known about the functions of cyclins in legume plants, especially Medicago truncatula. Our genome-wide analysis identified 58, 103, and 51 cyclin members in the M. truncatula, Glycine max, and Phaseolus vulgaris genomes. Phylogenetic analysis suggested that these cyclins could be classified into 10 types, and the CycB-like types (CycBL1-BL8) were the specific subgroups in M. truncatula, which was one reason for the expansion of the B-type in M. truncatula. All putative cyclin genes were mapped onto their own chromosomes of each genome, and 9 segmental duplication gene pairs involving 20 genes were identified in M. truncatula cyclins. Determined by quantitative real-time PCR, the expression profiling suggested that 57 cyclins in M. truncatula were differentially expressed in 9 different tissues, while a few genes were expressed in some specific tissues. Using the publicly available RNAseq data, the expression of Mtcyclins in the wild-type strain A17 and three nodule mutants during rhizobial infection showed that 23 cyclins were highly upregulated in the nodulation (Nod) factor-hypersensitive mutant sickle (skl) mutant after 12 h of rhizobium inoculation. Among these cyclins, six cyclin genes were also specifically expressed in roots and nodules, which might play specific roles in the various phases of Nod factor-mediated cell cycle activation and nodule development. Our results provide information about the cyclin gene family in legume plants, serving as a guide for further functional research on plant cyclins.

Genome-Wide Characterization, Evolution, and Expression Analysis of the Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Gene Family in Medicago truncatula.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) constitute the largest subfamily of receptor-like kinases (RLKs) in plants. They play roles in plant growth and developmental and physiological processes, but less is known about the functions of LRR-RLKs in Medicago truncatula. Our genome-wide analysis revealed 329 LRR-RLK genes in the M.truncatula genome. Phylogenetic and classification analysis suggested that these genes could be classified into 15 groups and 24 subgroups. A total of 321 genes were mapped onto all chromosomes, and 23 tandem duplications (TDs) involving 56 genes were distributed on each chromosome except 4. Twenty-seven M.truncatula LRR-RLK segmental duplication gene pairs were colinearly related. The exon/intron organization, motif composition and arrangements were relatively conserved among members of the same groups or subgroups. Using publicly available RNAseq data and quantitative real-time polymerase chain reaction (qRT-PCR), expression profiling suggested that LRR-RLKs were differentially expressed among different tissues, while some were expressed specifically in the roots and nodules. The expression of LRR-RLKs in A17 and 4 nodule mutants under rhizobial infection showed that 36 LRR-RKLs were highly upregulated in the sickle (skl) mutant [an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant] after 12 h of rhizobium inoculation. Among these LRR-RLKs, six genes were also expressed specifically in the roots and nodules, which might be specific to the Nod factor and involved in autoregulation of the nodulation signal. Our results provide information on the LRR-RLK gene family in M. truncatula and serve as a guide for functional research of the LRR-RLKs.

The lre-miR159a-LrGAMYB pathway mediates resistance to grey mould infection in Lilium regale.

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 H2 O2 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.

Analysis of WRKY transcription factors and characterization of two Botrytis cinerea-responsive LrWRKY genes from Lilium regale.

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.

Evaluation of resistance to Botrytis elliptica in Lilium hybrid cultivars.

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.

Transcriptome-Wide Analysis of Botrytis elliptica Responsive microRNAs and Their Targets in Lilium Regale Wilson by High-Throughput Sequencing and Degradome Analysis.

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.

Lilium spp. pollen in China (Liliaceae): taxonomic and phylogenetic implications and pollen evolution related to environmental conditions.

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.

Molecular phylogeny and genetic variation in the genus Lilium native to China based on the internal transcribed spacer sequences of nuclear ribosomal DNA.

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.

Identification of legume RopGEF gene families and characterization of a Medicago truncatula RopGEF mediating polar growth of root hairs.

Root hairs play important roles in the interaction of plants with their environment. Root hairs anchor the plant in the soil, facilitate nutrient uptake from the rhizosphere, and participate in symbiotic plant-microbe interactions. These specialized cells grow in a polar fashion which gives rise to their elongated shape, a process mediated in part by a family of small GTPases known as Rops. RopGEFs (GEF, guanine nucleotide exchange factor) activate Rops to effect tip growth in Arabidopsis pollen and root hairs, but the genes mediating tip growth in legumes have not yet been characterized. In this report we describe the Rop and RopGEF gene families from the model legume Medicago truncatula and from the crop legume soybean. We find that one member of the M. truncatula gene family, MtRopGEF2, is required for root hair development because silencing this gene by RNA interference affects the cytosolic Ca2+ gradient and subcellular structure of root hairs, and reduces root hair growth. Consistent with its role in polar growth, we find that a GFP::MtRopGEF2 fusion protein localizes in the apex of emerging and actively growing root hairs. The amino terminus of MtRopGEF2 regulates its ability to interact with MtRops in yeast, and regulates its biological activity in vivo.