Turgor regulation defect 1 proteins play a conserved role in pollen tube reproductive innovation of the angiosperms.

Sexual reproduction in angiosperms is siphonogamous, and the interaction between pollen tube and pistil is critical for successful fertilization. Our previous study demonstrated that mutation of the Arabidopsis turgor regulation defect 1 (TOD1) gene leads to reduced male fertility, a result of retarded pollen tube growth in the pistil. TOD1 encodes a Golgi-localized alkaline ceramidase, a key enzyme for the production of sphingosine-1-phosphate (S1P), which is involved in the regulation of turgor pressure in plant cells. However, whether TOD1s play a conserved role in the innovation of siphonogamy is largely unknown. In this study, we provide evidence that OsTOD1, which is similar to AtTOD1, is also preferentially expressed in rice pollen grains and pollen tubes. OsTOD1 knockout results in reduced pollen tube growth potential in rice pistil. Both the OsTOD1 genomic sequence with its own promoter and the coding sequence under the AtTOD1 promoter can partially rescue the attod1 mutant phenotype. Furthermore, TOD1s from other angiosperm species can partially rescue the attod1 mutant phenotype, while TOD1s from gymnosperm species are not able to complement the attod1 mutant phenotype. Our data suggest that TOD1 acts conservatively in angiosperms, and this opens up an opportunity to dissect the role of sphingolipids in pollen tube growth in angiosperms.

Transcriptomic and proteomic analysis reveals mechanisms of low pollen-pistil compatibility during water lily cross breeding.

BACKGROUND: In water lily (Nymphaea) hybrid breeding, breeders often encounter non-viable seeds, which make it difficult to transfer desired or targeted genes of different Nymphaea germplasm. We found that pre-fertilization barriers were the main factor in the failure of the hybridization of Nymphaea. The mechanism of low compatibility between the pollen and stigma remains unclear; therefore, we studied the differences of stigma transcripts and proteomes at 0, 2, and 6 h after pollination (HAP). Moreover, some regulatory genes and functional proteins that may cause low pollen-pistil compatibility in Nymphaea were identified. RESULTS: RNA-seq was performed for three comparisons (2 vs 0 HAP, 6 vs 2 HAP, 6 vs 0 HAP), and the number of differentially expressed genes (DEGs) was 8789 (4680 were up-regulated), 6401 (3020 were up-regulated), and 11,284 (6148 were up-regulated), respectively. Using label-free analysis, 75 (2 vs 0 HAP) proteins (43 increased and 32 decreased), nine (6 vs 2 HAP) proteins (three increased and six decreased), and 90 (6 vs 0 HAP) proteins (52 increased and 38 decreased) were defined as differentially expressed proteins (DEPs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed that the DEGs and DEPs were mainly involved in cell wall organization or biogenesis, S-adenosylmethionine (SAM) metabolism, hydrogen peroxide decomposition and metabolism, reactive oxygen species (ROS) metabolism, secondary metabolism, secondary metabolite biosynthesis, and phenylpropanoid biosynthesis. CONCLUSIONS: Our transcriptomic and proteomic analysis highlighted specific genes, incuding those in ROS metabolism, biosynthesis of flavonoids, SAM metabolism, cell wall organization or biogenesis and phenylpropanoid biosynthesis that warrant further study in investigations of the pollen-stigma interaction of water lily. This study strengthens our understanding of the mechanism of low pollen-pistil compatibility in Nymphaea at the molecular level, and provides a theoretical basis for overcoming the pre-fertilization barriers in Nymphaea in the future.

Morphophysiological dormancy in the basal angiosperm order Nymphaeales.

Background and Aims: Substantial evidence supports the hypothesis that morphophysiological dormancy (MPD) is the basal kind of seed dormancy in the angiosperms. However, only physiological dormancy (PD) is reported in seeds of the ANA-grade genus Nymphaea. The primary aim of this study was to determine the kind of dormancy in seeds of six species of Nymphaea from the wet-dry tropics of Australia. Methods: The effects of temperature, light and germination stimulants on germination were tested on multiple collections of seeds of N. immutabilis, N. lukei, N. macrosperma, N. ondinea, N. pubescens and N. violacea. Embryo growth prior to hypocotyl emergence was monitored. Key Results: Germination was generally <10 % after 28 d in control treatments. Germination percentage was highest at 30 or 35 °C for seeds exposed to light and treated with ethylene or in anoxic conditions in sealed vials of water, and it differed significantly between collections of N. lukei, N. macrosperma and N. violacea. Seeds of N. pubescens did not germinate under any of the conditions. Embryo growth (8-37 % in length) occurred before hypocotyl emergence (germination) in seeds of the five species that germinated. Conclusions: Fresh seeds were dormant, and the amount of pregermination embryo growth in seeds of N. lukei and N. immutabilis was relatively small, while in seeds of N. macrosperma, N. ondinea and N. violacea it was relatively large. Thus, seeds of N. lukei and N. immutabilis had PD and those of N. macrosperma, N. ondinea and N. violacea had MPD. Overall, we found that seeds in the most phylogenetically derived clades within Nymphaea have MPD, suggesting that PD is the most likely basal trait within the Nymphaeales. This study also highlights the broad range of dormancy types and germination strategies in the ANA-grade angiosperms.

Auxin controls circadian flower opening and closure in the waterlily.

BACKGROUND: Flowers open at sunrise and close at sunset, establishing a circadian floral movement rhythm to facilitate pollination as part of reproduction. By the coordination of endogenous factors and environmental stimuli, such as circadian clock, photoperiod, light and temperature, an appropriate floral movement rhythm has been established; however, the underlying mechanisms remain unclear. RESULTS: In our study, we use waterlily as a model which represents an early-diverging grade of flowering plants, and we aim to reveal the general mechanism of flower actions. We found that the intermediate segment of petal cells of waterlily are highly flexible, followed by a circadian cell expansion upon photoperiod stimuli. Auxin causes constitutively flower opening while auxin inhibitor suppresses opening event. Subsequent transcriptome profiles generated from waterlily's intermediate segment of petals at different day-time points showed that auxin is a crucial phytohormone required for floral movement rhythm via the coordination of YUCCA-controlled auxin synthesis, GH3-mediated auxin homeostasis, PIN and ABCB-dependent auxin efflux as well as TIR/AFB-AUX/IAA- and SAUR-triggered auxin signaling. Genes involved in cell wall organization were downstream of auxin events, resulting in the output phenotypes of rapid cell expansion during flower opening and cell shrinkage at flower closure stage. CONCLUSIONS: Collectively, our data demonstrate a central regulatory role of auxin in floral movement rhythm and provide a global understanding of flower action in waterlily, which could be a conserved feature of angiosperms.

Disentangling historical signal and pollinator selection on the micromorphology of flowers: an example from the floral epidermis of the Nymphaeaceae.

The family Nymphaeaceae includes most of the diversity among the ANA-grade angiosperms. Among the species of this family, floral structures and pollination strategies vary. The genus Victoria, as well as subgenera Lotos and Hydrocallis in Nymphaea, present night-blooming, scented flowers pollinated by scarab beetles. Such similar pollination strategies have led to macromorphological similarities among the flowers of these species, which could be interpreted as homologies or convergences based on different phylogenetic hypotheses about the relationships of these groups. We employed scanning electron microscopy of floral epidermis for seven species of the Nymphaeaceae with contrasting pollination biology to identify the main characters of the floral organs and the potential homologous nature of the structures involved in pollinator attraction. Moreover, we used transmission electron microscopy to observe ultrastructure of papillate-conical epidermis in the stamen of Victoria cruziana. We then tested the phylogenetic or ecological distribution of these traits using both consensus network approaches and ancestral state reconstruction on fixed phylogenies. Our results show that the night-blooming flowers present different specialisations in their epidermis, with V. cruziana presenting the most elaborate floral anatomy. We also identify for the first time the presence of conical-papillate cells in the order Nymphaeales. The epidermal characters tend to reflect phylogenetic relationships more than convergence due to pollinator selection. These results point to an independent and parallel evolution of scarab pollination in Nymphaeaceae and demonstrate the promise of floral anatomy as a phylogenetic marker. Moreover, they indicate a degree of sophistication in the anatomical basis of cantharophilous flowers in the Nymphaeales that diverges from the most simplistic views of floral evolution in the angiosperms.

Seasonal variations and aeration effects on water quality improvements and physiological responses of Nymphaea tetragona Georgi.

Seasonal variations and aeration effects on water quality improvements and the physiological responses of Nymphaea tetragona Georgi were investigated with mesocosm experiments. Plants were hydroponically cultivated in six purifying tanks (aerated, non-aerated) and the characteristics of the plants were measured. Water quality improvements in purifying tanks were evaluated by comparing to the control tanks. The results showed that continuous aeration affected the plant morphology and physiology. The lengths of the roots, petioles and leaf limbs in aeration conditions were shorter than in non-aeration conditions. Chlorophyll and soluble protein contents of the leaf limbs in aerated tanks decreased, while peroxidase and catalase activities of roots tissues increased. In spring and summer, effects of aeration on the plants were less than in autumn. Total nitrogen (TN) and ammonia nitrogen (NH4(+)-N) in aerated tanks were lower than in non-aerated tanks, while total phosphorus (TP) and dissolved phosphorus (DP) increased in spring and summer. In autumn, effects of aeration on the plants became more significant. TN, NH4(+)-N, TP and DP became higher in aerated tanks than in non-aerated tanks in autumn. This work provided evidences for regulating aeration techniques based on seasonal variations of the plant physiology in restoring polluted stagnant water.

The role of thermogenesis in the pollination biology of the Amazon waterlily Victoria amazonica.

BACKGROUND AND AIMS: Several families of tropical plants have thermogenic flowers that show a 2-d protogynous sequence. Most are pollinated by large beetles that remain for the entire period in the flowers, where they compete for mates and feed. Active beetles require high body temperatures that they can achieve endogenously at great energy expense or attain passively and cheaply in a warm environment. Floral heating is therefore hypothesized to be a direct energy reward to endothermic beetles, in addition to its accepted role in enhancing scent production. METHODS: This study measures the pattern of floral heat production (as temperature in 20 flowers and respiration rates in five flowers) in Victoria amazonica at field sites in Guyana and correlates floral temperatures with body temperatures necessary for activity in visiting Cyclocephala hardyi beetles. KEY RESULTS: Thermogenesis occurred in a bimodal pattern, with peaks associated with the arrival and departure of beetles near sunset. Peak CO(2) production rates averaged 2.9 micromol s(-1), equivalent to a heat production of 1.4 W. Heat was generated mainly in the floral chamber on the first evening and by the stamen complex on the second. Mean chamber temperature remained between 29.3 and 34.7 degrees C during the first night, when ambient temperature was 23.5-25.2 degrees C. Beetles actively competed for mates and consumed stylar processes in the floral chamber, where their mean thoracic temperature was 33.2 degrees C. At the lower ambient temperatures outside of the flower, beetles capable of sustained flight had a similar mean temperature of 32.0 degrees C. CONCLUSIONS: Floral heating is not only associated with attraction, but continues throughout the night when beetles are active inside the flower and increases again when they leave. Floral chamber temperatures similar to activity temperatures of actively endothermic beetles imply that thermogenesis is an energy reward.

Genetically based polymorphisms in morphology and life history associated with putative host races of the water lily leaf beetle, Galerucella nymphaeae.

A host race is a population that is partially reproductively isolated from other conspecific populations as a direct consequence of adaptation to a specific host. The initial step in host race formation is the establishment of genetically based polymorphisms in, for example, morphology, preference, or performance. In this study we investigated whether polymorphisms observed in Galerucella nymphaeae have a genetic component. Galerucella nymphaeae, the water lily leaf beetle, is a herbivore which feeds and oviposits on the plant hosts Nuphar lutea and Nymphaea alba (both Nymphaeaceae) and Rumex hydrolapathum and Polygonum amphibium (both Polygonaceae). A full reciprocal crossing scheme (16 crosses, each replicated 10 times) and subsequent transplantation of 1,001 egg clutches revealed a genetic basis for differences in body length and mandibular width. The heritability value of these traits, based on midparent-offspring regression, ranged between 0.53 and 0.83 for the different diets. Offspring from Nymphaeaceae parents were on average 12% larger and had on average 18% larger mandibles than offspring from Polygonaceae parents. Furthermore, highly significant correlations were found between feeding preference of the offspring and the feeding preference of their parents. Finally, two fitness components were measured: development time and survival. Development time was influenced by diet, survival both by cross type and diet, the latter of which suggest adaptation of the beetles. This suggestion is strengthened by a highly significant cross x diet interaction effect for development time as well as for survival, which is generally believed to indicate local adaptation. Although no absolute genetic incompatibility among putative host races was observed, survival of the between-host family offspring, on each diet separately, was lower than the survival of the within-host family offspring on that particular host. Survival of offspring of two Nymphaeaceae parents was about two times higher on Nymphaeaceae than on Polygonaceae, whereas survival of offspring of two Polygonaceae parents was 11 times higher on Polygonaceae than on Nymphaeaceae (based on untransformed data). Based on these results, we conclude that genetically determined polymorphisms in morphology and feeding preference exist in G. nymphaeae, resulting in differential performance. Furthermore, in each diet separately, offspring of between-host family crosses were less fit than offspring of within-host family crosses. These results support the hypothesis that within this species two host races can be distinguished.