Integrating Multi-Omics Analysis Reveals the Regulatory Mechanisms of White-Violet Mutant Flowers in Grape Hyacinth (Muscari latifolium).

Grape hyacinth (Muscari spp.) is a famous bulbous blue flower; however, few bicolor varieties are available in the market. Therefore, the discovery of bicolor varieties and understanding of their mechanisms are crucial to the breeding of new varieties. In this study, we report a significant bicolor mutant with white upper and violet lower portions, with both parts belonging to a single raceme. Ionomics showed that pH and metal element contents were not responsible for the bicolor formation. Targeted metabolomics illustrated that the content of the 24 color-related compounds was significantly lower in the upper part than that in the lower part. Moreover, full-length transcriptomics combined with second-generation transcriptomics revealed 12,237 differentially expressed genes in which anthocyanin synthesis gene expression of the upper part was noted to be significantly lower than that of the lower part. Transcription factor differential expression analysis was used to describe the presence of a pair of MaMYB113a/b sequences, with low levels of expression in the upper part and high expression in the lower part. Furthermore, tobacco transformation confirmed that overexpression of MaMYB113a/b can promote anthocyanin accumulation in tobacco leaves. Accordingly, the differential expression of MaMYB113a/b contributes the formation of a bicolor mutant in Muscari latifolium.

Investigation of the phytochemical composition and remedial effects of southern grape hyacinth (Muscari neglectum Guss. ex Ten.) plant extract against carbon tetrachloride-induced oxidative stress in rats.

We aimed to determine the phytochemical contents of the aerial part M. neglectum aerial part (MAP) and M. neglectum bulb (MB) ethanolic extract of Muscari neglectum and to investigate their protective effects on gastric damage induced by carbon tetrachloride (CCl4) in rats. After the toxicity testing, 42 female Wistar albino rats were divided into 7 groups, Control, MAP, MB, CCl4, CCl4 + MAP, CCl4 + MB, and CCl4 + Silymarin groups. At the end of the experiment, the serum biochemical parameters, antioxidant defense enzymes, and malondialdehyde (MDA) contents in the stomach tissue were evaluated to determine the antioxidant role of the M. neglectum extracts. According to the gas chromatography-mass spectroscopy, fatty acid analysis, octadecadienoic, and 9,12,15 octadecatrienoic fatty acids were found as major fatty acids in the MAP, whereas 9,12 octadecadienoic and octadecanoic acids were the major fatty acids in the MB. According to the liquid chromatography-tandem mass spectrometry, quinic acid, fumaric acid, gentisic acid, caffeic acid, kaempferol, and apigenin were found in the MAP, while quinic acid, fumaric acid, caffeic acid, and kaempferol were found in the MB. The total phenolic and flavonoid contents in the extract were determined in the MAP and MB. The MAP and MB extracts generally caused a statistically significant decrease in the MDA content and increase in the antioxidant parameters in the stomach tissue. It was concluded that MAP and MB extracts may have antioxidant and gastric protective effects due to the phytochemical content of M. neglectum.HighlightsAccording to LC-MS/MS results, quinic acid, fumaric acid, chemferol, apigenin, and caffeic acid were determined as major compounds in M. neglectum extracts.According to GC-MS results, octadecadienoic, octadecatrienoic, and octadecanoic methyl esters were the major fatty acids of the M. neglectum extracts.The M. neglectum extracts regulated the levels of stomach damage and biochemical parameters.The M. neglectum extracts extract might have pharmaceutical-nutritional potential.

The R2R3MYB transcription factors MaMYBF and MaMYB1 regulate flavonoid biosynthesis in grape hyacinth.

R2R3 MYBs play vital roles in the regulation of flavonoid biosynthesis. However, the regulatory network of R2R3 MYBs in flavonoid biosynthesis is not fully understood in grape hyacinth (Muscari spp.). Here, we identified two R2R3 MYBs, MaMYBF and MaMYB1, as potential regulators of flavonol and anthocyanin biosynthesis, respectively. MaMYBF and MaMYB1 expression was elevated during flower development and was light-induced, and the expression patterns were related to those of the flavonoid structural genes MaFLS and MaDFR, respectively. The BiFC assay verified that MaMYB1 interacts with MabHLH1, but MaMYBF does not. A dual luciferase assay revealed that MaMYBF alone strongly activated pMaFLS, and its activation was attenuated at reduced doses of MaMYBF in the presence of MabHLH1, MaMybA, and MaMYB1. MaDFR transcription mediated by MaMybA and MabHLH1 was inhibited by MaMYB1. Moreover, overexpression of MaMYBF and MaMYB1 in tobacco reduced flower pigmentation and repressed the expression of flavonoid pathway key structural genes. Therefore, MaMYBF regulates the flavonol pathway independently of cofactors. Whereas MaMYB1 regulates anthocyanin biosynthesis by binding to MabHLH1 and disrupting the MaMybA-bHLH complex in grape hyacinth. Our results offer new insights into the intricate regulatory network of flavonoids in grape hyacinth involving the regulation of both flavonol and anthocyanin.

Two B-Box Proteins, MaBBX20 and MaBBX51, Coordinate Light-Induced Anthocyanin Biosynthesis in Grape Hyacinth.

Floral colour is an important agronomic trait that influences the commercial value of ornamental plants. Anthocyanins are a class of flavonoids and confer diverse colours, and elucidating the molecular mechanisms that regulate their pigmentation could facilitate artificial manipulation of flower colour in ornamental plants. Here, we investigated the regulatory mechanism of light-induced anthocyanin biosynthesis during flower colouration in grape hyacinth (Muscari spp.). We studied the function of two B-box proteins, MaBBX20 and MaBBX51. The qPCR revealed that MaBBX20 and MaBBX51 were associated with light-induced anthocyanin biosynthesis. Both MaBBX20 and MaBBX51 are transcript factors and are specifically localised in the nucleus. Besides, overexpression of MaBBX20 in tobacco slightly increased the anthocyanin content of the petals, but reduced in MaBBX51 overexpression lines. The yeast one-hybrid assays indicated that MaBBX20 and MaBBX51 did not directly bind to the MaMybA or MaDFR promoters, but MaHY5 did. The BiFC assay revealed that MaBBX20 and MaBBX51 physically interact with MaHY5. A dual luciferase assay further confirmed that the MaBBX20-MaHY5 complex can strongly activate the MaMybA and MaDFR transcription in tobacco. Moreover, MaBBX51 hampered MaBBX20-MaHY5 complex formation and repressed MaMybA and MaDFR transcription by physically interacting with MaHY5 and MaBBX20. Overall, the results suggest that MaBBX20 positively regulates light-induced anthocyanin biosynthesis in grape hyacinth, whereas MaBBX51 is a negative regulator.

Single measurement detection of individual cell ionic oscillations using an n-type semiconductor - electrolyte interface.

Pollen tubes are used as models in studies on the type of tip-growth in plants. They are an example of polarised and rapid growth because pollen tubes are able to quickly invade the flower pistil in order to accomplish fertilisation. How different ionic fluxes are perceived, processed or generated in the pollen tube is still not satisfactorily understood. In order to measure the H+, K+, Ca2+ and Cl- fluxes of a single pollen tube, we developed an Electrical Lab on a Photovoltaic-Chip (ELoPvC) on which the evolving cell was immersed in an electrolyte of a germination medium. Pollen from Hyacinthus orientalis L. was investigated ex vivo. We observed that the growing cell changed the (redox) potential in the medium in a periodic manner. This subtle measurement was feasible due to the effects that were taking place at the semiconductor-liquid interface. The experiment confirmed the existence of the ionic oscillations that accompany the periodic extension of pollen tubes, thereby providing - in a single run - the complete discrete frequency spectrum and phase relationships of the ion gradients and fluxes, while all of the metabolic and enzymatic functions of the cell life cycle were preserved. Furthermore, the global 1/fα characteristic of the power spectral density, which corresponds to the membrane channel noise, was found.

Invasive floating macrophytes reduce greenhouse gas emissions from a small tropical lake.

Floating macrophytes, including water hyacinth (Eichhornia crassipes), are dominant invasive organisms in tropical aquatic systems, and they may play an important role in modifying the gas exchange between water and the atmosphere. However, these systems are underrepresented in global datasets of greenhouse gas (GHG) emissions. This study investigated the carbon (C) turnover and GHG emissions from a small (0.6 km(2)) water-harvesting lake in South India and analysed the effect of floating macrophytes on these emissions. We measured carbon dioxide (CO2) and methane (CH4) emissions with gas chambers in the field as well as water C mineralization rates and physicochemical variables in both the open water and in water within stands of water hyacinths. The CO2 and CH4 emissions from areas covered by water hyacinths were reduced by 57% compared with that of open water. However, the C mineralization rates were not significantly different in the water between the two areas. We conclude that the increased invasion of water hyacinths and other floating macrophytes has the potential to change GHG emissions, a process that might be relevant in regional C budgets.

Bioethanol production from sodium hydroxide/hydrogen peroxide-pretreated water hyacinth via simultaneous saccharification and fermentation with a newly isolated thermotolerant Kluyveromyces marxianu strain.

In this study, bioethanol production from NaOH/H2O2-pretreated water hyacinth was investigated. Pretreatment of water hyacinth with 1.5% (v/v) H2O2 and 3% (w/v) NaOH at 25 °C increased the production of reducing sugars (223.53 mg/g dry) and decreased the cellulose crystallinity (12.18%), compared with 48.67 mg/g dry and 22.80% in the untreated sample, respectively. The newly isolated Kluyveromyces marxianu K213 showed greater ethanol production from glucose (0.43 g/g glucose) at 45 °C than did the control Saccharomyces cerevisiae angel yeast. The maximum ethanol concentration (7.34 g/L) achieved with K. marxianu K213 by simultaneous saccharification and fermentation (SSF) from pretreated water hyacinth at 42 °C was 1.78-fold greater than that produced by angel yeast S. cerevisiae at 30 °C. The present work demonstrates that bioethanol production achieved via SSF of NaOH/H2O2-pretreated water hyacinth with K. marxianu K213 is a promising strategy to utilize water hyacinth biomass.

Isotope geochemistry. Biological signatures in clumped isotopes of O2.

The abundances of molecules containing more than one rare isotope have been applied broadly to determine formation temperatures of natural materials. These applications of "clumped" isotopes rely on the assumption that isotope-exchange equilibrium is reached, or at least approached, during the formation of those materials. In a closed-system terrarium experiment, we demonstrate that biological oxygen (O2) cycling drives the clumped-isotope composition of O2 away from isotopic equilibrium. Our model of the system suggests that unique biological signatures are present in clumped isotopes of O2­and not formation temperatures. Photosynthetic O2 is depleted in (18)O(18)O and (17)O(18)O relative to a stochastic distribution of isotopes, unlike at equilibrium, where heavy-isotope pairs are enriched. Similar signatures may be widespread in nature, offering new tracers of biological and geochemical cycling.

Spatial and temporal localization of homogalacturonans in Hyacinthus orientalis L. ovule cells before and after fertilization.

KEY MESSAGE: The composition of homogalacturonans (HGs) in the ovule and the female gametophyte cell walls was shown to be rearranged dynamically during sexual reproduction of H. orientalis. In angiosperms, homogalacturonans (HGs) play an important role in the interaction between the male gametophyte and the pistil transmitting tract, but little is known about the participation of these molecules at the final stage of the progamic phase and fertilization. The aim of our study was to perform immunocytochemical localization of highly (JIM7 MAb) and weakly (JIM5 MAb) methyl esterified and Ca(2+)-associated HG (2F4 MAb) in the ovule and female gametophyte cells of Hyacinthus orientalis before and after fertilization. It was found that pollination induced the rearrangement of HG in (1) the micropylar canal of the ovule, (2) the filiform apparatus of the synergids, and (3) the region of fusion between sperm cells and their target cells. Fertilization led to further changes in pectin composition of these three regions of the ovule. A new cell wall was synthesized around the zygote with a characteristic pattern of localization of all examined HG fractions, which we called "sporoderm-like". The developing endosperm prepared for cellularization by synthesizing highly methyl-esterified HG, which was stored in the cytoplasm. Pollination- and fertilization-induced changes in the composition of the HG in the micropyle of the ovule and the apoplast of female gametophyte cells are discussed in the context of: (1) micropylar pollen tube guidance, (2) preparation of the egg cell and the central cells for fusion with sperm cells, and (3) the polyspermy block.

Effects of temperature and light intensity on growth and physiology in purple root water hyacinth and common water hyacinth (Eichhornia crassipes).

In this study, the interaction between temperature and light intensity was investigated in common water hyacinth (CWH) and purple root water hyacinth (PRWH). Effects of different temperatures (11/5, 18/11, 25/18, and 32/25 °C day/night) simultaneously applied at various light intensities (100, 300, and 600 µmol m(-2) s(-1)) to the plants were detected by measuring changes in the root lengths, protein content, sugar content, malondialdehyde (MDA) content, photosynthesis, and dissolved oxygen (DO). Temperature and light intensity significantly influence the growth of water hyacinths, and there was significant interaction among these environmental factors. The results suggest that several environmental factors act synergistically on the growth and physiology of water hyacinths. The higher new root length (NRL) in PRWH indicated that its root growth capacity is higher than in CWH. The soluble sugar content in leaves of CWH was higher than PRWH, indicating that relatively higher sugar content in CWH to low-temperature stress may support its tolerant nature. Lower temperature and light intensity can stimulate the accumulation of MDA content. The net photosynthetic rate (Pn) in leaves of CWH was higher than PRWH. In low temperature, increase light intensity can stimulate the Pn of PRWH and CWH. In CWH and PRWH, Pn showed a similar trend as noted for stomatal conductance (Cond) and transpiration rate (Tr). The capacity of PRWH in adding oxygen to the water column is better than those of CWH.

[Effect of hyacinth mulching on rice (Oryza sativa L.) uptake and utilization of nitrogen].

Nitrogen is the most important element for rice growth, and hyacinth can absorb large quantities of nitrogen and accumulate in their tissues. The field experiment was conducted to investigate the effects of hyacinth mulching on rice nitrogen (N) concentration, uptake, efficiency and allocation at different growth stages. Taked Yun 2645 as a material, the nitrogen uptake and utilization of rice were evaluated under two different levels of N input: low (LN, 120 kg x hm(-2)) and normal N (NN, 240 kg x hm(-2)) in 2009. Main results showed that: (1) Compared with AMB, hyacinth mulching significantly increased N concentration in rice plant over the season, hyacinth mulching significantly increased nitrogen accumulation in rice plant, and the increasing rate was gradual addition from mid-tillering to heading, but from heading to maturity the increasing rate was gradual decline. (2) Hyacinth mulching had no obvious effect on nitrogen allocation pattern in leaves and stems of rice over the season, significantly decreased nitrogen allocation pattern in spikes. (3) Except mid-tillering, hyacinth mulching resulted in the significant decrease in N use efficiency for biomass (NUEp) over the season and in N use efficiency for grain yield (NUEg) at grain maturity, but no effect was observed on nitrogen harvest index (NHI). (4) Nitrogen concentration and accumulation at most growth stages of rice increased with increasing N supply (p < 0.05 or 0.01), but NUEp and NUEg showed the opposite trends. 1(5) nteractions between hyacinth mulching x N were not observed for N uptake and utilization. Hyacinth mulching increased N concentration and N uptake, decreased NUEp and NUEg at most growth stages of rice.

[Purification effects of large-area planting water hyacinth on water environment of Zhushan Bay, Lake Taihu].

Using water hyacinth and other fast-growing and high biomass of floating plants to purify polluted water has become an efficient and effective ecological restoration method at present. Effects of nutrients adsorption and water purification of planting water hyacinth on water quality in Zhushan Bay were studied. The results indicated that no anoxia was observed in water hyacinth planting areas because of wave disturbance and strong water exchange. Concentrations of TN and TP in water hyacinth planting areas were higher than that in the outside of stocking area (the content ranged 3.03-7.45 mg/L and 0.15-0.38 mg/L, respectively), and the content changes ranged 3.37-8.02 mg/L and 0.15-0.36 mg/L,respectively. The higher concentration of TN and TP in water indicated the water body was heavily polluted. Water hyacinth roots have a strong ability to adsorb suspended solids and algae cells, the concentration of Chl-a in stocking areas was higher than that in stocking fringe and outside, the maximum Chlorophyll in the stocking region in August was 177.01 mg/m3, and at the same time the concentrations in planting fringe and outside were 101.53 mg/m3 and 76.96 mg/m, respectively. Higher Chl-a content on water hyacinth roots indicated that water hyacinth had strong blocking effects on algae cells, and demonstrated it had a great purification effects on eutrophicated water, and it also provides a basis for the larger polluted water bodies purification in using water hyacinth.

Nuclear activity of sperm cells during Hyacinthus orientalis L. in vitro pollen tube growth.

In this study, the transcriptional state and distribution of RNA polymerase II, pre-mRNA splicing machinery elements, and rRNA transcripts were investigated in the sperm cells of Hyacinthus orientalis L. during in vitro pollen tube growth. During the second pollen mitosis, no nascent transcripts were observed in the area of the dividing generative cell, whereas the splicing factors were present and their pools were divided between newly formed sperm cells. Just after their origin, the sperm cells were shown to synthesize new RNA, although at a markedly lower level than the vegetative nucleus. The occurrence of RNA synthesis was accompanied by the presence of RNA polymerase II and a rich pool of splicing machinery elements. Differences in the spatial pattern of pre-mRNA splicing factors localization reflect different levels of RNA synthesis in the vegetative nucleus and sperm nuclei. In the vegetative nucleus, they were localized homogenously, whereas in the sperm nuclei a mainly speckled pattern of small nuclear RNA with a trimethylguanosine cap (TMG snRNA) and SC35 protein distribution was observed. As pollen tube growth proceeded, inhibition of RNA synthesis in the sperm nuclei was observed, which was accompanied by a gradual elimination of the splicing factors. In addition, analysis of rRNA localization indicated that the sperm nuclei are likely to synthesize some pool of rRNA at the later steps of pollen tube. It is proposed that the described changes in the nuclear activity of H. orientalis sperm cells reflect their maturation process during pollen tube growth, and that mature sperm cells do not carry into the zygote the nascent transcripts or the splicing machinery elements.

Gummosis in grape hyacinth (Muscari armeniacum) bulbs: hormonal regulation and chemical composition of gums.

The purpose of this study was to investigate the hormonal regulation of gummosis in grape hyacinth (Muscari armeniacum) bulbs, focusing especially on the chemical composition of the gums. The application of ethephon (2-chloroethylphosphonic acid), an ethylene-releasing compound, at 1% and 2% (w/w) in lanolin as well as ethylene induced gummosis in the bulbs within several days. Methyl jasmonate (JA-Me, 0.1-2% in lanolin) alone had no effect on gummosis. However, simultaneous application of JA-Me and ethephon led to extreme stimulation of ethephon-induced gummosis. Ethephon-induced gummosis in the bulbs depended on the maturation stage of the bulbs, increasing from April to July, but decreasing from August to September. Regardless of the presence of JA-Me, the application of ethephon to the inflorescence axis of grape hyacinths did not induce gummosis. Gel permeation chromatography analysis revealed that gums were homogenous polysaccharides with an average molecular mass of ca. 8.3 kDa. Analysis of the sugar composition of the gums after hydrolysis revealed that the molar ratio of Rha:Ara:Gal:GalA:GlcA was 25:10:40:7:15. These results suggest that principal factors of gummosis as well as the chemical composition of gums differ between species of bulbous plants.

[Effects of large-area planting water hyacinth on macro-benthos community structure and biomass].

The effects on macro-benthos and benthos environment of planting 200 hm2 water hyacinth (E. crassipens) in Zhushan Bay, Lake Taihu, were studied during 8-10 months consecutive surveys. Results indicated that average densities of mollusca (the main species were Bellamya aeruginosa) in far-planting, near-planting and planting area were 276.67, 371.11 and 440.00 ind/m2, respectively, and biomass were 373.15, 486.57 and 672.54 g/m2, respectively, showed that average density and biomass of planting area's were higher than those of others. However, the average density and biomass of Oligochaeta (the main species was Limodrilus hoffmeisteri) and Chironomidae in planting area were lower than that of outside planting area. The density and biomass of three dominant species of benthic animal increased quickly during 8-9 months, decreased quickly in October inside and outside water hyacinth planting area. The reason of this phenomenon could be possible that lots of cyanobacteria cells died and consumed dissolve oxygen in proceed decomposing. Algae cells released lots of phosphorus and nitrogen simultaneously, so macro-benthos died in this environment. The indexes of Shannon-Weaver and Simpson indicated that water environment was in moderate polluted state. On the basis of the survey results, the large-area and high-density planting water hyacinth haven't demonstrated a great impact on macrobenthos and benthos environment in short planting time (about 6 months planting time).

Distribution of poly(A) RNA and splicing machinery elements in mature Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro.

The localization of poly(A) mRNA and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and the SC35 protein, in mature Hyacinthus orientalis L. pollen grains before anthesis and pollen tubes germinating in vitro were analyzed. The observations indicated a pattern of poly(A) mRNA distribution in mature pollen grains before anthesis which differed from that in germinating pollen grains. Directly before anthesis, poly(A) mRNA was homogeneously distributed throughout the whole cytoplasm, whereas after rehydration, it accumulated at one of the pollen poles. In the pollen tube, poly(A) mRNA was present in the cytoplasm, mainly in the areas beneath the cell membrane and the apical zone. Both before anthesis and during growth of the pollen tube, splicing snRNPs and SC35 protein were localized mainly in the area of the pollen nuclei. During anthesis and just after rehydration of the pollen grains, the pattern of labeling and the levels of the investigated antigens in the areas of the vegetative and generative nuclei were similar. During growth of the pollen tube, a change was observed in the distribution and an increase in the levels of trimethylguanosine snRNA and SC35 protein in the vegetative nucleus. Such a pattern of localization of the splicing machinery suggests resumption of transcription and/or maturation of pre-mRNA in the growing pollen tube.

Characterization and expression analysis of a MADS box gene, HoMADS2, in Hyacinthus orientalis L.

A MADS box gene HoMADS2 was cloned from Hyacinthus orientalis L. in this study. Sequence comparison revealed that HoMADS2 was highly homologous to the class B MADS box genes. Furthermore, phylogenetic analysis showed that HoMADS2 was closely related to PI gene family and this was also supported by the presence of specific diagnostic sites of PI homologs in K box domain and C terminal region,suggesting that HoMADS2 might be a PI-like gene. HoMADS2 mRNA was accumulated in all floral organs,different from the expression patterns of PI homologs in dicots. During in vitro flower development, HoMADS2 expression was constitutively expressed and not affected by the presence of cytokinin and auxin in the regenerated flowers. Our results indicated that the expression of HoMADS2 is different from those of both HAG1 and HoMADS1 in responding to plant hormones during in vitro flower development.