Metabolomics and transcriptome analysis of the biosynthesis mechanism of flavonoids in the seeds of Euryale ferox Salisb at different developmental stages.

Flavonoids belong to polyphenolic compounds, which are widely distributed in plants and have rich functions. Euryale ferox Salisb is an important medicinal and edible homologous plant, and flavonoids are its main functional substances. However, the biosynthesis mechanism of flavonoids in E. ferox is still poorly understood. To explore the dynamic changes of flavonoid biosynthesis during the development of E. ferox seeds, the targeted flavonoid metabolome was determined. A total of 129 kinds of flavonoid metabolites were characterized in the seeds of E. ferox, including 11 flavanones, 8 dihydroflavanols, 16 flavanols, 29 flavones, 3 isoflavones, 12 anthocyanins, 29 flavonols, 6 flavonoid carbonosides, 3 chalcones and 13 proanthocyanidins. The relative content of flavonoid metabolites accumulated continuously during the development of E. ferox seeds, and reached the highest at T30. In transcriptome, the expression of key genes in the flavonoid pathway, such as PAL, CHS, F3H, FLS, ANS, was highest in T30, which was consistent with the trend of metabolites. Six candidate transcription factors (R2R3MYBs and bHLHs) may affect the biosynthesis of flavonoids by regulating the expression of structural genes. Furthermore, transcriptome analysis and exogenous ABA and SA treatment demonstrated that ABA (PYR1, PP2Cs, SnRK2s) and SA (NPR1) are involved in the positive regulation of flavonoid biosynthesis. This study clarified the differential changes of flavonoid metabolites during the development of E. ferox seeds, confirmed that ABA and SA promote the synthesis of flavonoids, and found key candidate genes that are involved in the regulation of ABA and SA in the positive regulation of flavonoid biosynthesis.

Physiological and biochemical responses of Makhana (Euryale ferox) to gamma irradiation.

The impact of gamma irradiation on growth and physiology of Euryale ferox was described in the present investigation. E. ferox is an underutilized aquatic food crop that grows in shallow-water bodies in lower Assam regions and north Bihar of India. The seeds of E. ferox were irradiated with different doses of gamma irradiation ranging from 0 to 500 Gy. It was observed that the germination and survival percentage was inhibited by increasing the irradiation dose. However, plants developed from seed exposed to an irradiation dose beyond 100 Gy did not survive more than 1 month. Further growth parameters (leaf size and number, number of thorns, root number and length, and number of flower and seeds) were also compared with respect to non-irradiated plants. Physiological parameters, viz. chlorophyll a, chlorophyll b, total chlorophyll, photosynthetic rate, transpiration rate, stomatal conductance, and intracellular CO2 content was higher in the irradiation population of E. ferox. Superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were observed low in irradiated population of E. ferox. The proline and glycine betaine content was enhanced with increasing the irradiation dose. The present investigation explores the potential use of gamma rays in genetic improvement of E. ferox and improves understanding of the physiological responses inflicted by gamma irradiation.

Transcriptome sequencing and analysis during seed growth and development in Euryale ferox Salisb.

BACKGROUND: Euryale ferox Salisb., an annual aquatic plant, is the only species in the genus Euryale in the Nymphaeaceae. Seeds of E. ferox are a nutritious food and also used in traditional Chinese medicine (Qian Shi in Mandarin). The molecular events that occurred during seed development in E. ferox have not yet been characterized. In this study, we performed transcriptomic analysis of four developmental stages (T1, T2, T3, and T4) in E. ferox seeds with three biological replicates per developmental stage to understand the physiological and biochemical processes during E. ferox seeds development. RESULTS: 313,844,425 clean reads were assembled into 160,107 transcripts and 85,006 unigenes with N50 lengths of 2052 bp and 1399 bp, respectively. The unigenes were annotated using five public databases (NR, COG, Swiss-Prot, KEGG, and GO). In the KEGG database, all of the unigenes were assigned to 127 pathways, of which phenylpropanoid biosynthesis was associated with the synthesis of secondary metabolites during E. ferox seed growth and development. Phenylalanine ammonia-lyase (PAL) as the first key enzyme catalyzed the conversion of phenylalanine to trans-cinnamic acid, then was related to the synthesis of flavonoids, lignins and alkaloid. The expression of PAL1 reached its peak at T3 stage, followed by a slight decrease at T4 stage. Cytochrome P450 (P450), encoded by CYP84A1 (which also called ferulate-5-hydroxylase (F5H) in Arabidopsis), was mainly involved in the biosynthesis of lignins. CONCLUSIONS: Our study provides a transcriptomic analysis to better understand the morphological changes and the accumulation of medicinal components during E. ferox seed development. The increasing expression of PAL and P450 encoded genes in phenylpropanoid biosynthesis may promote the maturation of E. ferox seed including size, color, hardness and accumulation of medicinal components.

Evidence for parent-of-origin effects and interparental conflict in seeds of an ancient flowering plant lineage.

Theoretical and empirical studies have long connected the evolutionary innovation of endosperm, a genetically biparental product of a double fertilization process unique to flowering plants (angiosperms), to conflicting parental interests over offspring provisioning. Yet, none of these studies examined interparental conflict in representatives of any of the most ancient angiosperm lineages. We performed reciprocal interploidy crosses in the water lily Nymphaea thermarum, a member of one of the most ancient angiosperm lineages, Nymphaeales. We find that an excess of paternal genomes is associated with an increase in endosperm growth. By contrast, maternal ploidy negatively influences development or growth of all seed components, regardless of paternal genome dosage. Most relevant to the conflict over distribution of maternal resources, however, is that growth of the perisperm (seed storage tissue derived from the maternal sporophyte, found in all Nymphaeales) is unaffected by paternal genome dosage-ensuring maternal control of maternal resources. We conclude that the evolutionary transfer of embryo-nourishing function from a genetically biparental endosperm to a genetically maternal perisperm can be viewed as an effective maternal strategy to recapture control of resource distribution among progeny, and thus that interparental conflict has influenced the evolution of seed development in this ancient angiosperm lineage.

Economy, efficiency, and the evolution of pollen tube growth rates.

PREMISE: Pollen tube growth rate (PTGR) is an important aspect of male gametophyte performance because of its central role in the fertilization process. Theory suggests that under intense competition, PTGRs should evolve to be faster, especially if PTGR accurately reflects gametophyte quality. Oddly, we know remarkably little about how effectively the work of tube construction is translated to elongation (growth and growth rate). Here we test the prediction that pollen tubes grow equally efficiently by comparing the scaling of wall production rate (WPR) to PTGR in three water lilies that flower concurrently: Nymphaea odorata, Nuphar advena and Brasenia schreberi. METHODS: Single-donor pollinations on flower or carpel pairs were fixed just after pollen germination (time A) and 45 min later (time B). Mean PTGR was calculated as the average increase in tube length over that growth period. Tube circumferences (C) and wall thicknesses (W) were measured at time B. For each donor, WPR = mean (C × W) × mean PTGR. KEY RESULTS: Within species, pollen tubes maintained a constant WPR to PTGR ratio, but species had significantly different ratios. N. odorata and N. advena had similar PTGRs, but for any given PTGR, they had the lowest and highest WPRs, respectively. CONCLUSIONS: We showed that growth rate efficiencies evolved by changes in the volume of wall material used for growth and in how that material was partitioned between lateral and length dimensions. The economics of pollen tube growth are determined by tube design, which is consequent on trade-offs between efficient growth and other pollen tube functions.

Pollen structure and development in Nymphaeales: insights into character evolution in an ancient angiosperm lineage.

PREMISE OF THE STUDY: A knowledge of pollen characters in early-diverging angiosperm lineages is essential for understanding pollen evolution and the role of pollen in angiosperm diversification. In this paper, we report and synthesize data on mature pollen and pollen ontogeny from all genera of Nymphaeales within a comparative, phylogenetic context and consider pollen evolution in this early-diverging angiosperm lineage. We describe mature pollen characters for Euryale, Barclaya, and Nymphaea ondinea, taxa for which little to no structural data exist. METHODS: We studied mature pollen for all nymphaealean genera using light, scanning electron, and transmission electron microscopy. We reviewed published reports of nymphaealean pollen to provide a comprehensive discussion of pollen characters in water lilies. KEY RESULTS: Nymphaeales exhibit diversity in key pollen characters, including dispersal unit size, ornamentation, aperture morphology, and tapetum type. All Nymphaeales pollen are tectate-columellate, exhibiting one of two distinct patterns of infratectal ultrastructure-a thick infratectal space with robust columellae or a thin infratectal space with thin columellae. All genera have pollen with a lamellate endexine that becomes compressed in the proximal, but not distal wall. This endexine ultrastructure supports the operculate hypothesis for aperture origin. Nymphaeaceae pollen exhibit a membranous granular layer, which is a synapomorphy of the family. CONCLUSIONS: Variation in pollen characters indicates that significant potential for lability in pollen development was present in Nymphaeales at the time of its divergence from the rest of angiosperms. Structural and ontogenetic data are essential for interpreting pollen characters, such as infratectum and endexine ultrastructure in Nymphaeales.

Pollen tube development in two species of Trithuria (Hydatellaceae) with contrasting breeding systems.

Trithuria (Hydatellaceae; Nymphaeales) is unique among early-divergent angiosperms in that its species are extremely small and most have exceptionally short, annual life histories. Given the evolution of these extremes of size and development, we sought to understand whether post-pollination processes still varied predictably with breeding system in Trithuria. To address this question, we studied two Western Australian species, Trithuria austinensis (dioecious, obligately outcrossing) and Trithuria submersa (bisexual, highly selfing). To document developmental timing, carpels were hand-pollinated, collected at sequential time points, and examined with light and fluorescence microscopy. In both species, pollen tubes first entered ovules<1 h after pollination, but the pollen tube pathway of outcrossing T. austinensis was almost four times longer and its pollen tube growth rates were up to six times faster (≤2,166 vs. 321 µm/h) than those of T. submersa. T. austinensis also exhibited greater male investment, slower pollen germination, and greater pollen tube attrition. These differences in male gametophyte development are predicted for outcrossers versus selfers in phylogenetically derived angiosperms. These new data for Hydatellaceae reinforce the idea that an acceleration of pollen tube development occurred in the Nymphaeales stem lineage, before the origin of Hydatellaceae. We infer that a recent evolutionary transition to selfing in T. submersa has been accompanied by predictable modifications to reproductive development, which, because of the ancient relationship between Hydatellaceae and all other angiosperms, suggests that traits underlying the lability of flowering plant post-pollination biology were present early in their history.

Cabomba as a model for studies of early angiosperm evolution.

BACKGROUND: The angiosperms, or flowering plants, diversified in the Cretaceous to dominate almost all terrestrial environments. Molecular phylogenetic studies indicate that the orders Amborellales, Nymphaeales and Austrobaileyales, collectively termed the ANA grade, diverged as separate lineages from a remaining angiosperm clade at a very early stage in flowering plant evolution. By comparing these early diverging lineages, it is possible to infer the possible morphology and ecology of the last common ancestor of the extant angiosperms, and this analysis can now be extended to try to deduce the developmental mechanisms that were present in early flowering plants. However, not all species in the ANA grade form convenient molecular-genetic models. SCOPE: The present study reviews the genus Cabomba (Nymphaeales), which shows a range of features that make it potentially useful as a genetic model. We focus on characters that have probably been conserved since the last common ancestor of the extant flowering plants. To facilitate the use of Cabomba as a molecular model, we describe methods for its cultivation to flowering in the laboratory, a novel Cabomba flower expressed sequence tag database, a well-adapted in situ hybridization protocol and a measurement of the nuclear genome size of C. caroliniana. We discuss the features required for species to become tractable models, and discuss the relative merits of Cabomba and other ANA-grade angiosperms in molecular-genetic studies aimed at understanding the origin of the flowering plants.

Unique stigmatic hairs and pollen-tube growth within the stigmatic cell wall in the early-divergent angiosperm family Hydatellaceae.

BACKGROUND AND AIMS: The ultrastructure of the pollen tubes and the unusual multicellular stigmatic hairs of Trithuria, the sole genus of Hydatellaceae, are described in the context of comparative studies of stigmatic and transmitting tissue in other early-divergent angiosperms. METHODS: Scanning and transmission electron microscopy and immunocytochemistry are used to study the structure and composition of both mature and immature stigmatic hair cells and pollen-tube growth in Trithuria. KEY RESULTS: Trithuria possesses a dry-type stigma. Pollen tubes grow within the cell walls of the long multicellular stigmatic hairs. Immunocytochemistry results suggest that arabinogalactan proteins are involved in attracting the pollen tubes through the stigmatic cuticle. Most tubes grow along the hair axis towards its base, but some grow towards the hair apex, suggesting that pollen tubes are guided by both physical constraints such as microfibril orientation and the presence of binding factors such as unesterified pectins and adhesive proteins. CONCLUSIONS: The presence of a dry-type stigma in Trithuria supports the hypothesis that this condition is ancestral in angiosperms. Each multicellular stigmatic hair of Hydatellaceae is morphologically homologous with a stigmatic papilla of other angiosperms, but functions as an independent stigma and style. This unusual combination of factors makes Hydatellaceae a useful model for comparative studies of pollen-tube growth in early angiosperms.