Phytochemical Investigation and Reproductive Capacity of the Bulgarian Endemic Plant Species Marrubium friwaldskyanum Boiss. (Lamiaceae)

Marrubium friwaldskyanum Boiss (Lamiaceae) is a Bulgarian endemic species. Overall, the essential oil (EO) composition of M. friwaldskyanum was different from that of the other Marrubium species reported in the literature. The main EO constituents of M. friwaldskyanum were (E)-caryophyllene, germacrene D, and caryophyllene oxide. The effect of the harvest stage was significant only on α-copaene, (E)-caryophyllene, caryophyllene oxide, and τ-muurolol. The concentration of α-copaene (1.26–1.83% range of the total oil), (E)-caryophyllene (31–41%), caryophyllene oxide (6.4–11.8%), and τ-muurolol (1.3–2.8%) were the highest at 2–3 pair of leaves or before flowering and lower at flowering. The harvest stage did not significantly affect the concentrations of the other six identified EO compounds β-bourbonene (1.1%), α-humulene (2.8%), germacrene D (23.3%), bicyclogermacrene (2.85%), δ-cadinene (1.1%), and spathulenol (2.8%). In a separate experiment, grinding of the biomass prior to EO extraction had a significant effect only on the concentrations of D-limonene (0.24–3.3%) and bicyclogermacrene (3.6–9.1%). Grinding in water or without water, maceration, and addition of Tween®20 had rather small effects on the EO profile. The identified EO constituents and their mean concentrations in this experiment were (E)-caryophyllene (25.4%), germacrene D (17.6%), caryophyllene oxide (9.1%), spathulenol (6.5%), τ-muurolol (5.0%), carvacrol (3.9%), α-copaene (2.5%), β-bourbonene (2.5%), δ-cadinene (2.4%), α-humulene (1.8%), and Z-β-farnesene (1.3%). Embryological studies observed anther and the development of the male gametophyte and ovule and development of the female gametophyte of M. friwaldskyanum. Furthermore, pollen and seed viability assays were conducted, and mass spectrometry-based metabolomics analysis of an extract from shoots revealed the presence of 45 natural products, identified as flavonoids, phenolic acids, and (tri)terpenoids. Overall, the phytochemistry and some of the microscopic analyses distinguished this endemic species from other species in Marrubium.

Genomic Signatures of Sexual Selection on Pollen-Expressed Genes in Arabis alpina

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.

Critical role of H3K27 methylation in cell fate determination of two cell lineages in male gametophyte

During angiosperm male gametogenesis, microspores divide to produce a vegetative cell (VC) and a male germline (MG), each with a distinct cell fate. How the MG cell/VC fate is determined remains largely unknown. Here, we report that H3K27me3 is essential for VC fate commitment and H3K27me3 erasure contributes to the MG cell fate initiation in male gametophyte of Arabidopsis. The VC-targeted H3K27me3 erasure disturbed the VC development and resulted in the VC fate shifting towards a gamete destination, which suggests that MG cells require H3K27me3 erasure for triggering the gamete cell fate. Multi-omics and cytologic analysis confirmed the occurrence of extensive cell identity transition due to H3K27me3 erasure. Therefore, we experimentally confirm that the MG cell/VC fate is epigenetically regulated. The H3K27 methylation plays a critical role in the guidance of MG cell/VC fate determination for pollen fertility in Arabidopsis. Our work also provides new evidences for two previous hypotheses that the germline cell fate is specified by the differential distribution of yet unknown determinant, and VC maintains the microspore's default program, namely the H3K27me3 setting, whereas MG requires reprogramming.

A Decade of Pollen Phosphoproteomics

Angiosperm mature pollen represents a quiescent stage with a desiccated cytoplasm surrounded by a tough cell wall, which is resistant to the suboptimal environmental conditions and carries the genetic information in an intact stage to the female gametophyte. Post pollination, pollen grains are rehydrated, activated, and a rapid pollen tube growth starts, which is accompanied by a notable metabolic activity, synthesis of novel proteins, and a mutual communication with female reproductive tissues. Several angiosperm species (Arabidopsis thaliana, tobacco, maize, and kiwifruit) were subjected to phosphoproteomic studies of their male gametophyte developmental stages, mostly mature pollen grains. The aim of this review is to compare the available phosphoproteomic studies and to highlight the common phosphoproteins and regulatory trends in the studied species. Moreover, the pollen phosphoproteome was compared with root hair phosphoproteome to pinpoint the common proteins taking part in their tip growth, which share the same cellular mechanisms.

Transformation of Seed Non-Transmissible Hop Viroids in Nicotiana benthamiana Causes Distortions in Male Gametophyte Development

Viroids are small, non-coding, parasitic RNAs that promote developmental distortions in sensitive plants. We analyzed pollen of Nicotiana benthamiana after infection and/or ectopic transformation with cDNAs of citrus bark cracking viroid (CBCVd), apple fruit crinkle viroid (AFCVd) and potato spindle tuber viroid (PSTVd) variant AS1. These viroids were seed non-transmissible in N. benthamiana. All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced by approximately 3.6 × 103, 800 and 59 times in mature pollen of CBCVd, AFCVd and PSTVd infected N. benthamiana, respectively, in comparison to leaves. These results suggest similar elimination processes during male gametophyte development as in the Nicotiana tabacum we presented in our previous study. Mature pollen of N. benthamiana showed no apparent defects in infected plants although all three viroids induced strong pathological symptoms on leaves. While Nicotiana species have naturally bicellular mature pollen, we noted a rare occurrence of mature pollen with three nuclei in CBCVd-infected N. benthamiana. Changes in the expression of ribosomal marker proteins in AFCVd-infected pollen were detected, suggesting some changes in pollen metabolism. N. benthamiana transformed with 35S-driven viroid cDNAs showed strong symptoms including defects in pollen development. A large number of aborted pollen (34% and 62%) and a slight increase of young pollen grains (8% and 15%) were found in mature pollen of AFCVd and CBCVd transformants, respectively, in comparison to control plants (3.9% aborted pollen and 0.3% young pollen). Moreover, pollen grains with malformed nuclei or trinuclear pollen were found in CBCVd-transformed plants. Our results suggest that “forcing” overexpression of seed non-transmissible viroid led to strong pollen pathogenesis. Viroid adaptation to pollen metabolism can be assumed as an important factor for viroid transmissibility through pollen and seeds.

Качество мужского гаметофита гибридов томата в условиях повышенной температуры

The aim of the research was to study the effect of high temperature on the variability of male gametophyte traits in hybrid F5 populations of tomato. Thus, it was found that the high temperature has a significant effect on the variability of the male gametophyte characteristics of tomato hybrids. For the combinations under study, it was revealed that the temperature factor and genotype are the main sources of variability of the male gametophyte traits against the background of elevated temperature. As a result of the experiments, the two most heat-resistant tomato genotypes were identified.

Влияние холодового стресса на некоторые признаки простых гибридов кукурузы

The purpose of this study was to determine the heritability of certain traits under low temperatures both at the stage of early plant development and at the haploid level, while identifying potentially stress resistant and productive hybrids. 14 simple hybrids of the first generation were used as the initial materi-al. The variability of signs of early development of plants and male gametophyte under low temperatures was evaluated. The coefficients of heritability of maternal and paternal genotypes under stressful condi-tions are calculated. A reliable dependence of the variability of the "pollen grain diameter" trait on the interaction of parental genotypes with a stress factor is shown. Hybrids Mo17xN6, Mo17xW23, A285xRf7, XL12xN6, XL12xP101 were the best in terms of the characteristics studied in the experiment.

ClCd and ClCf act redundantly at the TGN/EE and prevent acidification of the Golgi stack

The trans-Golgi network/early endosome (TGN/EE) serves as the central hub in which exo- and endocytic trafficking pathways converge and specificity of cargo routing needs to be achieved. Acidification is a hallmark of the TGN/EE and is maintained by the vacuolar H+-ATPase (V-ATPase) with support of proton-coupled antiporters. We show here that ClCd and ClCf, two distantly related members of the Arabidopsis chloride channel (ClC)-family co-localize in the TGN/EE, act redundantly, and are essential for male gametophyte development. Combining an inducible knock-down approach and in vivo pH-measurements, we show here that reduced ClC-activity does not affect pH in the TGN/EE but causes hyperacidification of trans-Golgi cisternae. Taken together, our results show that ClC-mediated anion transport into the TGN/EE is essential and affects spatio-temporal aspects of TGN/EE-maturation as well as its functional separation from the Golgi stack.

Genomic signatures of sexual selection on pollen-expressed genes in Arabis alpina

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.