Characterization of individual HIV-1 budding event using ultra-fast atomic force microscopy reveals a multiplexed role for VPS4

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Whereas the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (< 100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wildtype (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 30% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

Expression Analysis of DgD14, DgBRC1 and DgLsL in the Process of Chrysanthemum Lateral Bud Formation

The growth of lateral bud can greatly affect the development of apical bud and reduce the quality of single-flower cut chrysanthemum. However, the wide use of artificial bud removal in production leads to the increase on production cost. Therefore, it is important to study the lateral bud development mechanism in chrysanthemum for plant type regulation and genetic improvement. Auxin (IAA), cytokinins (CKs) and strigolactones (SLs) have direct or indirect effects on the formation of lateral buds. D14, BRC1 and LsL are key factors regulating the signal pathways of hormones, but their regulation mechanisms on the development of lateral buds in chrysanthemum are still unclear. In this study, single-flower cut chrysanthemum ‘Jinba’ and spray cut chrysanthemum ‘Fenyan’ were used as experimental materials. Quantitative real-time PCR was used to observe the effects of apical bud removal and exogenous hormones on the growth of lateral buds and the expression levels of DgD14, DgBRC1 and DgLsL, so as to clarify the expression characteristics of three genes in the process of lateral bud formation. The results showed that GA was effective in promoting the growth of lateral buds, whereas IAA and ABA had little effects on lateral bud growth or even inhibited. Removing apical dominance can significantly affect the expression levels of three genes, which regulated the formation and elongation of lateral buds. Additionally, the three genes showed different responses to different hormone treatments. DgD14 had a significant response to GA, but a gentle response to ABA. The expression levels of DgBRC1 varied in different trends, and it responded to IAA in a more dramatic way. The levels of DgLsL reached the peaks quickly before decreased in most experimental groups, and its response to GA was extraordinary severe.

Decapitation Experiments Combined with the Transcriptome Analysis Reveal the Mechanism of High Temperature on Chrysanthemum Axillary Bud Formation

Temperature is an important factor that largely affects the patterns of shoot branching in plants. However, the effect and mechanism of temperature on axillary bud development in chrysanthemum remains poorly defined. The purpose of the present study is to investigate the effect of high temperature on the axillary bud growth and the mechanism of axillary bud formation in chrysanthemum. Decapitation experiments combined with the transcriptome analysis were designed. Results showed that the axillary bud length was significantly inhibited by high temperature. Decapitation of primary shoot (primary decapitation) resulted in slower growth of axillary buds (secondary buds) under 35 °C. However, secondary decapitation resulted in complete arrest of tertiary buds at high temperature. These results demonstrated that high temperature not only inhibited axillary bud formation but also retarded bud outgrowth in chrysanthemum. Comparative transcriptome suggested differentially expressed gene sets and identified important modules associated with bud formation. This research helped to elucidate the regulatory mechanism of high temperature on axillary bud growth, especially bud formation in chrysanthemum. Meanwhile, in-depth studies of this imperative temperature signaling can offer the likelihood of vital future applications in chrysanthemum breeding and branching control.

The Effect of Supplementary LED Lighting on the Morphological and Physiological Traits of Miniature Rosa × Hybrida ‘Aga’ and the Development of Powdery Mildew (Podosphaera pannosa) under Greenhouse Conditions

We investigated the growth traits, flower bud formation, photosynthetic performance, and powdery mildew development in miniature Rosa × hybrida ‘Aga’ plants grown in the greenhouse under different light-emitting diode (LED) light spectra. Fluorescence-based sensors that detect the maximum photochemical efficiency of photosystem II (PS II) as well as chlorophyll and flavonol indices were used in this study. Five different LED light treatments as a supplement to natural sunlight with red (R), blue (B), white (W), RBW+FR (far-red) (high R:FR), and RBW+FR (low R:FR) were used. Control plants were illuminated only by natural sunlight. Plants were grown under different spectra of LED lighting and the same photosynthetic photon flux density (PPFD) (200 µmol m−2 s−1) at a photoperiod of 18 h. Plants grown under both RBW+FR lights were the highest, and had the greatest total shoot length, irrespective of R:FR. These plants also showed the highest maximum quantum yield of PS II (average 0.805) among the light treatments. Red monochromatic light and RBW+FR at high R:FR stimulated flower bud formation. Moreover, plants grown under red LEDs were more resistant to Podosphaera pannosa than those grown under other light treatments. The increased flavonol index in plants exposed to monochromatic blue light, compared to the W and control plants, did not inhibit powdery mildew development.

CONTENT OF PHOTOSYNTHETIC PIGMENTS IN ONTOGENESIS OF WILD AND CULTURAL PEA

The objective consisted in study of wild pea representatives on chlorophyll and carotenoid content for use in producing new varieties with high photosynthetic efficiency and optimal assimilate distribution. The research was conducted in 2016‑2017 in Federal Scientific Center of Legumes and Groat Crops (Orel). Plants were grown on experimental field with density 1.2 million plants/ha on triplicated plots 1 m2. Wild pea accessions k-5322 (asiaticum), k-3370 (elatius), k-4014 (elatius) from VIR collection and varieties Temp and Stabil were used. Wild accessions k-3370 and k-5322 had more chlorophyll a, chlorophyll b and carotenoids then leafy Temp variety on 24.5 %, 28.2 %, 41.5 % and 13.3 %, 2.7 %, 18.7 % respectively. Unlike the cultivated pea, accessions k-3370 and k-5322 retain high level of chlorophylls a and b in ontogenesis longer time. After transition from bud formation to the beginning of seed filling content of chlorophylls a and b in accession k-3370 was at the same level (11.71 mg/g и 11.1 mg/g respectively), in accession k-5322 it decreased insignificantly from 11.31 mg/g to 9.72 mg/g. Nevertheless, in variety Temp content of chlorophylls a and b significantly decreased on 34.1 % after transition from bud formation to the seed filling. Wild accession k-3370 like pea varieties had low chlorophyll a/b ratio (2.92), but accession k-5322 had the highest value (3.26) of the ratio. In wild accession k-3370 and k-5322 the chlorophyll a/b ratio was changed in ontogenesis that is no-typical for Temp variety. Pea wild accessions can be considered as sources of valuable alleles determining the high level of chlorophylls and carotenoids

Effects of carbon, nitrogen, phosphorus, and potassium on flowering and Fruiting of Glycyrrhiza uralensis

Carbon (C), nitrogen (N), phosphorus (P), and potassium (K) play an important role in flower bud differentiation and seed-filling; however, the effects of these elements on the flowering and fruiting of Glycyrrhiza uralensis Fisch. are not known. In this study, we evaluated the differences in the C, N, P, and K levels between the fruiting and nonfruiting plants of G. uralensis at different growth stages. The correlations between the elements C, N, P, and K and the flower and fruit falling rates, rate of empty seeds, rate of shrunken grains, and thousand kernel weight (TKW) were also determined. The results show that the P and K levels and C:N, P:N, and K:N ratios of flowering plants are significantly higher than those of nonflowering plants; N level of flowering plants is significantly lower than that of nonflowering plants at the flower bud differentiation stage. The number of inflorescences was positively correlated with C and K levels and C:N and K:N ratios. A low level of C, P, and K and high level of N in flowering and pod setting stage may lead to the flower and fruit drop of G. uralensis. The K level is significantly negatively correlated with the rates of empty and shrunken seeds. The N level is significantly positively correlated with TKW. Thus, high levels of C, P, and K might be beneficial to flower bud differentiation, while higher levels of N is not beneficial to the flower bud formation of G. uralensis. Higher levels of N and K at the filling stage were beneficial to the seed setting and seed-filling of G. uralensis.HighlightHigh levels of C, P, and K might be beneficial to flower bud differentiation, while higher levels of N is not beneficial to the flower bud formation of G. uralensis. Higher levels of N and K at the filling stage were beneficial to the seed setting and seed-filling of G. uralensis.

Composition and content of amino acids in plants of galega orientalis

The highest percentage of protein is observed at the beginning of the Bud formation phase, especially in the leaves. The amino acid composition of the protein does not change, but depending on the phase of development and analyze parts of the plant, the absolute content of their different.

Morpho-physiological reactions of gravisensitivity and adaptation to UV-radiation of the moss Bryum Caespiticiumhedw. from Antarctica

The adaptive physiological reactions of the moss Bryum caespiticium Hedw. from Antarctica to the influence of UV radiation and gravimorphoses as a factor of adaptive plasticity, associated with environmental conditions, were studied. As a control, B. caespiticium plants were collected in the Nature Reserve “Roztochchia” (Lviv region). In investigations, we used a sterile laboratory culture of mosses grown under controlled conditions in a phytotron. Moss shoots were irradiated with UV rays generated by an ultraviolet lamp OSRAM with an intensity of 4 kW/m2, which caused 50 % inhibition of plant regeneration (ED50). Physiological parameters were determined 24 h after exposure to UV radiation. The influence of gravity on the morphological form of B. caespiticium gametophyte turf and the interaction of light and gravity in gravi-/phototropism as a manifestation of gravimorphoses adaptability were analyzed. One of the objectives was to investigate the formation of gravimorphoses as a result of the initiation of cells’ branching processes and the formation of gametophore buds and to evaluate their role in the life cycle of B. caespiticium under extreme conditions. For this, we determined the branching coefficient of the gravitropic protonema, the inclination angle of the branches and the buds’ development depending on the interaction of photo- and gravitropism, under the influence of red and blue light, and the effect of UV on gravisensitivity. The influence of physiologically active red and blue light on the branching activity and bud formation on the gravitropic protonema of the Antarctic moss B. caespiticium was investigated. It was found that red light mainly inhibited graviperception and gravitropic growth of protonemata cells, resulting in a change of the response to gravity, but initiated high branching activity and, accordingly, another morphological form of turf. After the influence of the blue light, intensive bud formation and gametophore development were observed. Thus, gravitation promoted morphological variability and changes in the functional activity of cells at the juvenile stage of the protonemata development, which is important for the survival of the moss under extreme environmental conditions. After UV irradiation the gravisensitivity of the B. caespiticium protonemata decreased. However, due to the resistance of the moss sample from Antarctica to the prolonged influence of UV rays, gravitropic growth was not completely blocked, as in plants from the Lviv region. The effect of the ultraviolet irradiation on the antioxidant activity, the content of soluble (vacuolar) and cell wall-bound fractions of UV-absorbing phenolic components, flavonoids content and their absorption spectra, as well as the amount of carotenoids and anthocyanins in B. caespiticium shoots, were determined. It was established that B. caespiticium plants from Antarctica have 1.5 times higher antioxidant activity compared to plants from the Lviv region, which confirms the high level of protection against oxidative damage. UV irradiation activates the synthesis of UV-absorbing phenolic compounds in mosses. The shoots of B. caespiticium from Antarctica defined a higher content of phenols compared to samples from the Lviv region and their significant increase under the influence of UV radiation. The content of UV-absorbing compounds bound with the cell wall was higher than the concentration of soluble phenolic compounds, both in plants from Antarctica and in samples from the Lviv region, which indicates their participation in the mechanisms of cells protection from UV radiation. It was shown that the influence of UV irradiation induced an increase of flavonoids’ content in the shoots of both samples of B. caespiticium, but for plants from Antarctica, the concentration of flavonoids after stress was 1.7 times higher than in plants from the Lviv region. The absorption spectra of flavonoids revealed flavonols rutin and quercetin and flavone luteolin in both samples of B. caespiticium, which provide effective cells absorption of UV rays. The higher content of anthocyanins and carotenoids in moss shoots from Antarctica both in the control sample and after the exposure to UV radiation promotes the protection against damage and formation of the adaptive potential.

Androgen action in cell fate and communication during prostate development at single-cell resolution

Androgens/androgen receptor (AR) mediated signaling pathways are essential for prostate development, morphogenesis, and regeneration. Specifically, stromal AR-signaling has been shown to be essential for prostatic initiation. However, the molecular mechanisms underlying AR-initiated mesenchymal-epithelial interactions in prostate development remain unclear. Here, using a newly generated mouse model, we directly addressed the fate and role of genetically marked AR-expressing cells during embryonic prostate development. Androgen signaling-initiated signaling pathways were identified in mesenchymal niche populations at single cell transcriptomic resolution. The dynamic cell-signaling networks regulated by stromal AR were characterized in regulating prostatic epithelial bud formation. Pseudotime analyses further revealed the differentiation trajectory and fate of AR-expressing cells in both prostatic mesenchymal and epithelial cell populations. Specifically, the cellular properties of Zeb1-expressing progenitors were assessed. Selective deletion of AR signaling in a subpopulation mesenchymal rather than epithelial cells dysregulates the expression of the master regulators and significantly impairs prostatic bud formation. These data provide novel, high-resolution evidence demonstrating the important role of mesenchymal androgen signaling as cellular niches controlling prostate early development by initiating dynamic mesenchyme-epithelia cell interactions.