Lipidomic and transcriptomic profiles of glycerophospholipid metabolism during Hemerocallis citrina Baroni flowering.

BACKGROUND: Hemerocallis citrina Baroni (daylily) is a horticultural ornamental plant and vegetable with various applications as a raw material in traditional Chinese medicine and as a flavouring agent. Daylily contains many functional substances and is rich in lecithin, which is mostly composed of glycerophospholipids. To study the comprehensive dynamic changes in glycerophospholipid during daylily flowering and the underlying signalling mechanisms, we performed comprehensive, time-resolved lipidomic and transcriptomic analyses of 'Datong Huanghua 6' daylily. RESULTS: Labelling with PKH67 fluorescent antibodies clearly and effectively helped visualise lipid changes in daylily, while relative conductivity and malonaldehyde content detection revealed that the early stages of flowering were controllable processes; however, differences became non-significant after 18 h, indicating cellular damage. In addition, phospholipase D (PLD) and lipoxygenase (LOX) activities increased throughout the flowering process, suggesting that lipid hydrolysis and oxidation had intensified. Lipidomics identified 558 lipids that changed during flowering, with the most different lipids found 12 h before and 12 h after flowering. Transcriptome analysis identified 13 key functional genes and enzymes in the glycerophospholipid metabolic pathway. The two-way orthogonal partial least squares analysis showed that diacylglycerol diphosphate phosphatase correlated strongly and positively with phosphatidic acid (PA)(22:0/18:2), PA(34:2), PA(34:4), and diacylglycerol(18:2/21:0) but negatively with phospholipase C. In addition, ethanolamine phosphotransferase gene and phospholipid-N-methyltransferase gene correlated positively with phosphatidylethanolamine (PE)(16:0/18:2), PE(16:0/18:3), PE(33:2), and lysophosphatidylcholine (16:0) but negatively with PE(34:1). CONCLUSIONS: Overall, this study elucidated changes in the glycerophospholipid metabolism pathway during the daylily flowering process, as well as characteristic genes, thus providing a basis for future studies of glycerophospholipids and signal transduction in daylilies.

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.

High frequency in vitro regeneration of adventitious shoots in daylilies (Hemerocallis sp) stem tissue using thidiazuron.

BACKGROUND: Daylilies are a lucrative crop used for its floral beauty, medicinal proprieties, landscaping, fire prevention, nutritional value, and research. Despite the importance, daylilies remain extremely challenging for multiplying in vitro. The response difficulty is exacerbated because a few good protocols for daylilies micropropagation are generally difficult to reproduce across genotypes. An efficient strategy, currently applied at Langston University, is to systematically explore individual tissues or organs for their potential to micropropagation. This article is a partial report of the investigation carried out under room environmental conditions and focuses on developing an efficient daylilies in vitro propagation protocol that uses the stem tissue as the principal explant. RESULTS: In less than three months, using thidiazuron, the use of the stem tissue as the in vitro experimental explant was successful in inducing multiple shoots several folds greater than current daylilies shoot organogenesis protocols. The study showed that tissue culture can be conducted successfully under unrestricted room environmental conditions as well as under the controlled environment of a growth chamber. It also showed that splitting lengthwise stem explants formed multiple shoots several folds greater than cross-sectioned and inverted explants. Shoot conversion rate was mostly independent of the number of shoots formed per explants. The overall response was explant and genotype-dependent. Efficient responses were observed in all thidiazuron treatments. CONCLUSION: An efficient protocol, which can be applied for mass multiple shoots formation using the daylilies stem tissue as the main explant, was successfully developed. This could lead to a broad and rapid propagation of the crop under an array of environmental conditions to meet the market demand and hasten exogenous gene transfer and breeding selection processes.

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.

Photoinhibition-like damage to the photosynthetic apparatus in plant leaves induced by submergence treatment in the dark.

Submergence is a common type of environmental stress for plants. It hampers survival and decreases crop yield, mainly by inhibiting plant photosynthesis. The inhibition of photosynthesis and photochemical efficiency by submergence is primarily due to leaf senescence and excess excitation energy, caused by signals from hypoxic roots and inhibition of gas exchange, respectively. However, the influence of mere leaf-submergence on the photosynthetic apparatus is currently unknown. Therefore, we studied the photosynthetic apparatus in detached leaves from four plant species under dark-submergence treatment (DST), without influence from roots and light. Results showed that the donor and acceptor sides, the reaction center of photosystem II (PSII) and photosystem I (PSI) in leaves were significantly damaged after 36 h of DST. This is a photoinhibition-like phenomenon similar to the photoinhibition induced by high light, as further indicated by the degradation of PsaA and D1, the core proteins of PSI and PSII. In contrast to previous research, the chlorophyll content remained unchanged and the H2O2 concentration did not increase in the leaves, implying that the damage to the photosynthetic apparatus was not caused by senescence or over-accumulation of reactive oxygen species (ROS). DST-induced damage to the photosynthetic apparatus was aggravated by increasing treatment temperature. This type of damage also occurred in the anaerobic environment (N2) without water, and could be eliminated or restored by supplying air to the water during or after DST. Our results demonstrate that DST-induced damage was caused by the hypoxic environment. The mechanism by which DST induces the photoinhibition-like damage is discussed below.

Relative role of flower color and scent on pollinator attraction: experimental tests using F1 and F2 hybrids of daylily and nightlily.

The daylily (Hemerocallis fulva) and nightlily (H. citrina) are typical examples of a butterfly-pollination system and a hawkmoth-pollination system, respectively. H. fulva has diurnal, reddish or orange-colored flowers and is mainly pollinated by diurnal swallowtail butterflies. H. citrina has nocturnal, yellowish flowers with a sweet fragrance and is pollinated by nocturnal hawkmoths. We evaluated the relative roles of flower color and scent on the evolutionary shift from a diurnally flowering ancestor to H. citrina. We conducted a series of experiments that mimic situations in which mutants differing in either flower color, floral scent or both appeared in a diurnally flowering population. An experimental array of 6 × 6 potted plants, mixed with 24 plants of H. fulva and 12 plants of either F1 or F2 hybrids, were placed in the field, and visitations of swallowtail butterflies and nocturnal hawkmoths were recorded with camcorders. Swallowtail butterflies preferentially visited reddish or orange-colored flowers and hawkmoths preferentially visited yellowish flowers. Neither swallowtail butterflies nor nocturnal hawkmoths showed significant preferences for overall scent emission. Our results suggest that mutations in flower color would be more relevant to the adaptive shift from a diurnally flowering ancestor to H. citrina than that in floral scent.

Reproductive isolation on interspecific backcross of F1 pollen to parental species, Hemerocallis fulva and H. citrina (Hemerocallidaceae).

Reproductive failure of backcross could play important roles in determining the evolutionary outcome of hybridization. However, such studies have been somewhat fewer than those of the F1-producing cross and F1 x F1 cross. We conducted hand-pollination backcross experiments using Hemerocallis fulva, H. citrina, and their F1 hybrids. Seed set per flower of the backcrosses reduced to about 50% of the control cross, irrespective of ovule parent species. This symmetrical seed set reduction on the backcross might be caused by the death of backcross embryo as a result of Dobzhansky-Muller incompatibility by recessive alleles.

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.