Widely-targeted metabolomics and transcriptomics identify metabolites associated with flowering regulation of Choy Sum.

Choy Sum, a stalk vegetable highly valued in East and Southeast Asia, is characterized by its rich flavor and nutritional profile. Metabolite accumulation is a key factor in Choy Sum stalk development; however, no research has focused on metabolic changes during the development of Choy Sum, especially in shoot tip metabolites, and their effects on growth and flowering. Therefore, in the present study, we used a widely targeted metabolomic approach to analyze metabolites in Choy Sum stalks at the seedling (S1), bolting (S3), and flowering (S5) stages. In total, we identified 493 metabolites in 31 chemical categories across all three developmental stages. We found that the levels of most carbohydrates and amino acids increased during stalk development and peaked at S5. Moreover, the accumulation of amino acids and their metabolites was closely related to G6P, whereas the expression of flowering genes was closely related to the content of T6P, which may promote flowering by upregulating the expressions of BcSOC1, BcAP1, and BcSPL5. The results of this study contribute to our understanding of the relationship between the accumulation of stem tip substances during development and flowering and of the regulatory mechanisms of stalk development in Choy Sum and other related species.

Codon usage characterization and phylogenetic analysis of the mitochondrial genome in Hemerocallis citrina.

BACKGROUND: Hemerocallis citrina Baroni is a traditional vegetable crop widely cultivated in eastern Asia for its high edible, medicinal, and ornamental value. The phenomenon of codon usage bias (CUB) is prevalent in various genomes and provides excellent clues for gaining insight into organism evolution and phylogeny. Comprehensive analysis of the CUB of mitochondrial (mt) genes can provide rich genetic information for improving the expression efficiency of exogenous genes and optimizing molecular-assisted breeding programmes in H. citrina. RESULTS: Here, the CUB patterns in the mt genome of H. citrina were systematically analyzed, and the possible factors shaping CUB were further evaluated. Composition analysis of codons revealed that the overall GC (GCall) and GC at the third codon position (GC3) contents of mt genes were lower than 50%, presenting a preference for A/T-rich nucleotides and A/T-ending codons in H. citrina. The high values of the effective number of codons (ENC) are indicative of fairly weak CUB. Significant correlations of ENC with the GC3 and codon counts were observed, suggesting that not only compositional constraints but also gene length contributed greatly to CUB. Combined ENC-plot, neutrality plot, and Parity rule 2 (PR2)-plot analyses augmented the inference that the CUB patterns of the H. citrina mitogenome can be attributed to multiple factors. Natural selection, mutation pressure, and other factors might play a major role in shaping the CUB of mt genes, although natural selection is the decisive factor. Moreover, we identified a total of 29 high-frequency codons and 22 optimal codons, which exhibited a consistent preference for ending in A/T. Subsequent relative synonymous codon usage (RSCU)-based cluster and mt protein coding gene (PCG)-based phylogenetic analyses suggested that H. citrina is close to Asparagus officinalis, Chlorophytum comosum, Allium cepa, and Allium fistulosum in evolutionary terms, reflecting a certain correlation between CUB and evolutionary relationships. CONCLUSIONS: There is weak CUB in the H. citrina mitogenome that is subject to the combined effects of multiple factors, especially natural selection. H. citrina was found to be closely related to Asparagus officinalis, Chlorophytum comosum, Allium cepa, and Allium fistulosum in terms of their evolutionary relationships as well as the CUB patterns of their mitogenomes. Our findings provide a fundamental reference for further studies on genetic modification and phylogenetic evolution in H. citrina.

Integration of mRNA and miRNA Analysis Reveals the Post-Transcriptional Regulation of Salt Stress Response in Hemerocallis fulva.

MicroRNAs (miRNAs) belong to non-coding small RNAs which have been shown to take a regulatory function at the posttranscriptional level in plant growth development and response to abiotic stress. Hemerocallis fulva is an herbaceous perennial plant with fleshy roots, wide distribution, and strong adaptability. However, salt stress is one of the most serious abiotic stresses to limit the growth and production of Hemerocallis fulva. To identify the miRNAs and their targets involved in the salt stress resistance, the salt-tolerant H. fulva with and without NaCl treatment were used as materials, and the expression differences of miRNAs-mRNAs related to salt-tolerance were explored and the cleavage sites between miRNAs and targets were also identified by using degradome sequencing technology. In this study, twenty and three significantly differential expression miRNAs (p-value < 0.05) were identified in the roots and leaves of H. fulva separately. Additionally, 12,691 and 1538 differentially expressed genes (DEGs) were also obtained, respectively, in roots and leaves. Moreover, 222 target genes of 61 family miRNAs were validated by degradome sequencing. Among the DE miRNAs, 29 pairs of miRNA targets displayed negatively correlated expression profiles. The qRT-PCR results also showed that the trends of miRNA and DEG expression were consistent with those of RNA-seq. A gene ontology (GO) enrichment analysis of these targets revealed that the calcium ion pathway, oxidative defense response, microtubule cytoskeleton organization, and DNA binding transcription factor responded to NaCl stress. Five miRNAs, miR156, miR160, miR393, miR166, and miR396, and several hub genes, squamosa promoter-binding-like protein (SPL), auxin response factor 12 (ARF), transport inhibitor response 1-like protein (TIR1), calmodulin-like proteins (CML), and growth-regulating factor 4 (GRF4), might play central roles in the regulation of NaCl-responsive genes. These results indicate that non-coding small RNAs and their target genes that are related to phytohormone signaling, Ca2+ signaling, and oxidative defense signaling pathways are involved in H. fulva's response to NaCl stress.

Transcriptome analysis of response strategy in Hemerocallis fulva under drought stress.

BACKGROUND: Hemerocallis fulva is an important ground cover plant widely used in urban greening. The analysis of the molecular mechanism underlying the drought response of H. fulva can lay a foundation for improving its adaptability and expanding its planting area. OBJECTIVE: To reveal the drought response mechanisms of H. fulva, identify candidate unigenes associated with drought response, and lay a foundation for further unigenes functional study and drought resistance improvement of H. fulva via genetic engineering. METHODS: RNA was isolated from H. fulva under different experimental conditions. De novo transcriptomic analysis of the samples was performed to screen drought response unigenes. The transcriptional changes of candidate drought response unigenes were verified by quantitative real-time PCR. RESULTS: The differentially expressed unigenes and their functions were analyzed after H. fulva treated by PEG-simulated drought stress and rewatering. The candidate unigenes, associated with H. fulva drought response, were identified after transcriptome analysis. Then, the transcription level of drought response unigenes of H. fulva under different conditions was further verified. Abscisic acid, protein phosphorylation, sterol biosynthesis and ion transport were involved in drought response with quick restore in H. fulva. The response unigenes, involved in hormone (ABA, JA, CK and GA) signaling pathways, defense response, high light response, karrikin response and leaf shaping, can maintain at changed expression levels even after stress withdraw. CONCLUSION: Hemerocallis fulva has unique drought response mechanism. Negative regulation mechanism may play more important roles in drought response of H. fulva. The analysis of candidate unigenes, associated with drought response, lays a foundation for further drought resistance improvement of H. fulva.

Molecular Characterization and Expression Patterns of the HkSVP Gene Reveal Distinct Roles in Inflorescence Structure and Floral Organ Development in Hemerocallis fulva.

SHORT VEGETATIVE PHASE (SVP) genes are members of the well-known MADS-box gene family that play a key role in regulating vital developmental processes in plants. Hemerocallis are perennial herbs that exhibit continuous flowering development and have been extensively used in landscaping. However, there are few reports on the regulatory mechanism of flowering in Hemerocallis. To better understand the molecular basis of floral formation of Hemerocallis, we identified and characterized the SVP-like gene HkSVP from the Hemerocallis cultivar 'Kanai Sensei'. Quantitative RT-PCR (qRT-PCR) indicated that HkSVP transcript was mainly expressed in the vegetative growth stage and had the highest expression in leaves, low expression in petals, pedicels and fruits, and no expression in pistils. The HkSVP encoded protein was localized in the nucleus of Arabidopsis protoplasts and the nucleus of onion epidermal cells. Yeast two hybrid assay revealed that HKSVP interacted with Hemerocallis AP1 and TFL1. Moreover, overexpression of HkSVP in Arabidopsis resulted in delayed flowering and abnormal phenotypes, including enriched trichomes, increased basal inflorescence branches and inhibition of inflorescence formation. These observations suggest that the HkSVP gene may play an important role in maintaining vegetative growth by participating in the construction of inflorescence structure and the development of flower organs.

Morning and evening alarm of the circadian clock for flower opening times in Hemerocallis.

Previous studies demonstrated that flower opening time (FOT) is a stable trait and precisely controlled by a circadian clock responsive to the environment. It plays a vital role in improving fertility. Hemerocallis spp. has different FOTs divided into two types: nocturnal and diurnal. To explore the regulatory mechanisms of their FOTs, we carried out a transcriptome sequencing experiment at different developmental stages of an F1 population with different FOTs. 55,883 unigenes were obtained, and 9234 differential genes were identified. Co-expression was analyzed by K-means clustering and weighted gene co-expression network analysis. Results showed that after entering reproductive growth, two FOT types of Hemerocallis had increased expression of genes related to photosynthetic metabolism and sensitivity to environmental response such as light and hormone signal transmission. Circadian rhythm-related activities were enriched in hub genes during the flowering stage. Genes showing differential expression between the two Hemerocallis groups were related to environmental response and photosynthesis pathways. Putative circadian clock genes displayed differences in expression across the flower opening stage in both groups of Hemerocallis. Twenty-three key circadian clock genes were identified, which related to sensitivity to light signal input and gating. These genes might closely relate to FOTs in Hemerocallis.

Evolutionary history of Hemerocallis in Japan inferred from chloroplast and nuclear phylogenies and levels of interspecific gene flow.

The perennial herb genus Hemerocallis (Asphodelaceae) shows four flowering types: diurnal half-day, diurnal one-day, nocturnal half-day, and nocturnal one-day flowering. These flowering types are corresponding to their main pollinators, and probably act as a primary mechanism of reproductive isolation. To examine how the four flowering types diverged, we reconstructed the phylogeny of the Japanese species of Hemerocallis using 1615 loci of nuclear genome-wide SNPs and 2078 bp sequences of four cpDNA regions. We also examined interspecific gene flows among taxa by an Isolation-with-Migration model and a population structure analysis. Our study revealed an inconsistency between chloroplast and nuclear genome phylogenies, which may have resulted from chloroplast capture. Each of the following five clusters is monophyletic and clearly separated on the nuclear genome-wide phylogenetic tree: (I) two nocturnal flowering species with lemon-yellow flowers, H. citrina (half-day flowering) and H. lilioasphodelus (one-day flowering); (II) a diurnal one-day flowering species with yellow-orange flowers, H. middendorffii; (III) a variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. disticha; (IV) another variety of a diurnal half-day flowering species with reddish orange flowers, H. fulva var. aurantiaca, and a diurnal one-day flowering species with yellow-orange flowers, H. major; (V) a diurnal half-day flowering species with yellow-orange flowers, H. hakuunensis. The five clusters are consistent with traditional phenotype-based taxonomy (cluster I, cluster II, and clusters III-V correspond to Hemerocallis sect. Hemerocallis, Capitatae, and Fulvae, respectively). These findings could indicate that three flowering types (nocturnal flowering, diurnal one-day flowering, and diurnal half-day flowering) diverged in early evolutionary stages of Hemerocallis and subsequently a change from diurnal half-day flowering to diurnal one-day flowering occurred in a lineage of H. major. While genetic differentiation among the five clusters was well maintained, significant gene flow was detected between most pairs of taxa, suggesting that repeated hybridization played a role in the evolution of those taxa.

Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani.

Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the 'Datong' landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species.

Daylily protein constituents of the pollen and stigma a proteomics approach.

This study was aimed at the identification and quantification of the protein components of the pollen grains in parallel with the distal stigmatic tissue of tetraploid cultivars. Proteomes were analyzed using iTRAQ 4plex labeling, peptides separation by online RP-nano-LC and analysis by ESI-MS/MS. Protein identification and quantification were made using the Asparagales database as a reference. A total of 524,037 MS/MS spectra were produced from pollen and stigma samples. From these, a total of 8368 peptides wereidentified corresponding to 994 unique peptides and 432 protein groups. Among them, 128 differentially expressed proteins were retained for further analysis. In absence of the daylily genome availability, we exploited numerous databases and bioinformatics resources to exploring the putative biological functions of these proteins. The profile of differentially expressed proteins suggests an important representation of functions associated to the signalling and response against endogenous and environmental stresses, including several enzymes implicated in the biosynthesis of antibiotics. The abundance in stigma of several structural proteins of the ribosomal sub-units as well as of the core histones suggest that the translation processes and the regulation of gene expression in stigma is a more active mechanism than in pollen. In addition, pollen prioritizes the synthesis of fructose and glucose as opposed to sucrose in stigma as a source of energy. Finally, the modulated proteins in Hemerocallis point to several pathways that give potential clues concerning the molecular mechanisms underlying the functions of the pollen and the stigmatic fluid in daylily reproduction.

Difference in flowering time can initiate speciation of nocturnally flowering species.

Isolation mechanisms that prevent gene flow between populations prezygotically play important roles in achieving speciation. In flowering plants, the nighttime flowering system provides a mechanism for isolation from diurnally flowering species. Although this system has long been of interest in evolutionary biology, the evolutionary process leading to this system has yet to be elucidated because of the lack of good model species. However, the genetic mechanisms underlying the differences in flowering times and the traits that attract pollinators between a pair of diurnally and nocturnally flowering species have recently been identified in a few cases. This identification enables us to build a realistic model for theoretically studying the evolution of a nocturnally flowering species. In this study, based on previous experimental data, we assumed a model in which two loci control the flowering time and one locus determines a trait that attracts pollinators. Using this model, we evaluated the possibility of the evolution of a nocturnally flowering species from a diurnally flowering ancestor through simulations. We found that a newly emerging nighttime flowering flower exhibited a sufficiently high fitness, and the evolution of a nocturnally flowering species from a diurnally flowering species could be achieved when hybrid viability was intermediate to low, even in a completely sympatric situation. Our results suggest that the difference in flowering time can act as a magic trait that induces both natural selection and assortative mating and would play an important role in speciation between diurnally and nocturnally flowering species pairs.

Construction and significance analysis of the MicroRNA expression profile of Hemerocallis fulva at low temperature.

MicroRNA (miRNA) identification was performed in Hemerocallis fulva by high-throughput sequencing in combination with bioinformatics prediction. A total of 14,843,184 and 16,072,575 RNA sequences were explored under normal and low temperature conditions, respectively. There was a significant difference in RNAs species and quantity between the two samples. Of all the miRNAs, 26 were significantly upregulated and 30 were significantly downregulated, while nine were either significantly upregulated or downregulated under low-temperature stress. Twenty-one highly expressed miRNA families were screened in at least six species. The number of miRNA families was very similar between monocotyledons and dicotyledons, and only a few were more frequently found in monocotyledons.

Pollinator-mediated selection on flower color, flower scent and flower morphology of Hemerocallis: evidence from genotyping individual pollen grains on the stigma.

To trace the fate of individual pollen grains through pollination processes, we determined genotypes of single pollen grains deposited on Hemerocallis stigmas in an experimental mixed-species array. Hemerocallis fulva, pollinated by butterflies, has diurnal, reddish and unscented flowers, and H. citrina, pollinated by hawkmoths, has nocturnal, yellowish and sweet scent flowers. We observed pollinator visits to an experimental array of 24 H. fulva and 12 F2 hybrids between the two species (H. fulva and H. citrina) and collected stigmas after every trip bout of swallowtail butterflies or hawkmoths. We then measured selection by swallowtail butterflies or hawkmoths through male and female components of pollination success as determined by single pollen genotyping. As expected, swallowtail butterflies imposed selection on reddish color and weak scent: the number of outcross pollen grains acquired is a quadratic function of flower color with the maximum at reddish color, and the combined pollination success was maximal at weak scent (almost unrecognizable for human). This explains why H. fulva, with reddish flowers and no recognizable scent, is mainly pollinated by swallowtail butterflies. However, we found no evidence of hawkmoths-mediated selection on flower color or scent. Our findings do not support a hypothesis that yellow flower color and strong scent intensity, the distinctive floral characteristics of H. citrina, having evolved in adaptations to hawkmoths. We suggest that the key trait that triggers the evolution of nocturnal flowers is flowering time rather than flower color and scent.

Effects of population succession on demographic and genetic processes: predictions and tests in the daylily Hemerocallis thunbergii (Liliaceae).

Spatial genetic structure within plant populations is influenced by variation in demographic processes through space and time, including a population's successional status. To determine how demographic structure and fine-scale genetic structure (FSGS) change with stages in a population's successional history, we studied Hemerocallis thunbergii (Liliaceae), a nocturnal flowering and hawkmoth-pollinated herbaceous perennial with rapid population turnover dynamics. We examined nine populations assigned to three successive stages of population succession: expansion, maturation, and senescence. We developed stage-specific expectations for within-population demographic and genetic structure, and then for each population quantified the spatial aggregation of individuals and genotypes using spatial autocorrelation methods (nonaccumulative O-ring and kinship statistics, respectively), and at the landscape level measured inbreeding and genetic structure using Wright's F-statistics. Analyses using the O-ring statistic revealed significant aggregation of individuals at short spatial scales in expanding and senescing populations, in particular, which may reflect restricted seed dispersal around maternal individuals combined with relatively low local population densities at these stages. Significant FSGS was found for three of four expanding, no mature, and only one senescing population, a pattern generally consistent with expectations of successional processes. Although allozyme genetic diversity was high within populations (mean %P = 78.9 and H(E) = 0.281), landscape-level differentiation among sites was also high (F(ST) = 0.166) and all populations exhibited a significant deficit of heterozygotes relative to Hardy-Weinberg expectations (range F = 0.201-0.424, mean F(IS) = 0.321). Within populations, F was not correlated with the degree of FSGS, thus suggesting inbreeding due primarily to selfing as opposed to mating among close relatives in spatially structured populations. Our results demonstrate considerable variation in the spatial distribution of individuals and patterns and magnitude of FSGS in H. thunbergii populations across the landscape. This variation is generally consistent with succession-stage-specific differences in ecological processes operating within these populations.

Post-pollination reproductive isolation between diurnally and nocturnally flowering daylilies, Hemerocallis fulva and Hemerocallis citrina.

To examine whether floral and post-pollination isolation develops independently or not, we conducted a crossing experiment between Hemerocallis fulva and Hemerocallis citrina that shows large floral divergence adapted for diurnal and nocturnal pollinators that have been believed to be fully cross-fertile. Flowers of the two species from sympatric populations were hand-pollinated with conspecific pollen from the same population (control), interspecific pollen from the same area (sympatric cross), and interspecific pollen from the different area (allopatric cross). After capsule dehiscence, the fruit set, seed set per fruit and seed set per flower were determined among three cross categories. The seed sets per flower were 32 and 77% lower in sympatric and allopatric crosses than in the control when H. fulva was the pollen recipient. There was no difference in three reproductive measures among the cross categories when H. citrina was the pollen recipient. This finding indicates that post-pollination isolation does exist between H. fulva and H. citrina, although it is partial, asymmetric, and weakened in sympatry. Our result suggests that floral and post-pollination isolation may develop independently, and reinforcement may not be a general phenomenon in plants.

Bimodal distribution of flowering time in a natural hybrid population of daylily (Hemerocallis fulva) and nightlily (Hemerocallis citrina).

Time of flower anthesis in a day is thought to evolve in response to the time of pollinator activities. We studied blooming and withering time in natural populations of daylily (Hemerocallis fulva), nightlily (Hemerocallis citrina) and their hybrids, and also in an artificially obtained array of the F1 hybrids. Blooming time of H. fulva varied from 4:30 to 7:30 and H. citrina varied from 16:30 to 20:30. In a natural hybrid population, blooming time and withering time showed discontinuous bimodal distribution in spite that morphological traits of flowers showed continuous unimodal variation. Most F1 hybrids showed diurnal flowering. These findings indicate that only a few genes have strong phenotypic effect on the determination of flowering time in Hemerocallis, and suggest that the evolution from a H. fulva-like ancestor to H. citrina was not a continuous process by accumulation of minute mutations.