In Which Way Do the Flower Properties of the Specialist Orchid Goodyera repens Meet the Requirements of Its Generalist Pollinators?

This article is the next part of a series of studies documenting the influence of flower traits on the reproductive success (RS) of orchids. Knowledge of factors influencing RS helps to understand the mechanisms and processes crucial for shaping plant-pollinator interactions. The aim of the present study was to determine the role of flower structure and nectar composition in shaping the RS of the specialist orchid Goodyea repens, which is pollinated by generalist bumblebees. We found a high level of pollinaria removal (PR) and female reproductive success (fruiting, FRS) as well as a high level of variation between populations, although in certain populations pollination efficiency was low. Floral display traits, mainly inflorescence length, influenced FRS in certain populations. Among the flower traits, only the height of flowers was correlated with FRS in one population, suggesting that the flower structure of this orchid is well adapted to pollination by bumblebees. The nectar of G. repens is diluted and dominated by hexoses. Sugars were less important in shaping RS than amino acids. At the species level, twenty proteogenic and six non-proteogenic AAs were noted, along with their differentiated amounts and participation in particular populations. We found that distinct AAs or their groups mainly shaped PR, especially when correlations were considered at the species level. Our results suggest that both the individual nectar components and the ratios between them have an impact on G. repens RS. Because different nectar components influence the RS parameters in different ways (i.e., negatively or positively), we suggest that different Bombus species play the role of main pollinators in distinct populations.

Does Reproductive Success in Natural and Anthropogenic Populations of Generalist Epipactis helleborine Depend on Flower Morphology and Nectar Composition?

The purpose of our study was to determine the role of flower structure and nectar composition in shaping the reproductive success (RS) of the generalist orchid Epipactis helleborine in natural and anthropogenic populations. We supposed that the distinct character of two groups of habitats creates different conditions for plant-pollinator relationships, thus influencing reproductive success in E. helleborine populations. Both pollinaria removal (PR) and fruiting (FRS) were differentiated between the populations. On average, FRS was almost two times higher in the anthropogenic than in the natural populations. The difference between the two population groups in PR was smaller but still statistically significant. RS parameters were correlated with some floral display and flower traits. Floral display influenced RS only in three anthropogenic populations. Flower traits had a weak influence on RS (10 of the 192 cases analyzed). The more important trait in shaping RS was nectar chemistry. The nectar of E. helleborine is relatively diluted with a lower sugar concentration in the anthropogenic than in the natural populations. In the natural populations, domination of sucrose over hexoses was found, while in the anthropogenic populations, hexoses were more abundant and the participation of sugars was balanced. In some populations, sugars influenced RS. In E. helleborine nectar, 20 proteogenic and 7 non-proteogenic amino acids (AAs) were found with a clear domination of glutamic acid. We noted relationships between some AAs and RS, but distinct AAs shaped RS in different populations, and their impact was independent of their participation. Our results indicate that the flower structure and nectar composition of E. helleborine reflect its generalistic character and meet the requirements of a wide range of pollinators. Simultaneously, the differentiation of flower traits suggests a variation in pollinator assemblages in particular populations. Knowledge about the factors influencing RS in distinct habitats helps to understand the evolutionary potential of species and to understand mechanisms and processes crucial for shaping interactions between plants and pollinators.

Melatonin Involved in Protective Effects against Cadmium Stress in Wolffia arrhiza.

Melatonin (MT) is a new plant hormone that protects against adverse environmental conditions. In the present study, the responses of Wolffia arrhiza exposed to cadmium (Cd) and MT were analyzed. Quantitative analysis of MT and precursors of its biosynthesis was performed using LC-MS-MS. The photosynthetic pigments and phytochelatins (PCs) contents were determined using HPLC, while protein and monosaccharides, stress markers, and antioxidant levels were determined using spectrophotometric methods. Interestingly, the endogenous level of MT and its substrates in W. arrhiza exposed to 1-100 µM Cd was significantly higher compared to the control. Additionally, the application of 25 µM MT and Cd intensified the biosynthesis of these compounds. The most stimulatory effect on the growth and content of pigments, protein, and sugars was observed in plants treated with 25 µM MT. In contrast, Cd treatment caused a decrease in plant weight and level of these compounds, while the application of 25 µM MT mitigated the inhibitory effect of Cd. Additionally, Cd enhanced the level of stress markers; simultaneously, MT reduced their content in duckweed exposed to Cd. In plants treated with Cd, PC levels were increased by Cd treatment and by 25 µM MT. These results confirmed that MT mitigated the adverse effect of Cd. Furthermore, MT presence was reported for the first time in W. arrhiza. In summary, MT is an essential phytohormone for plant growth and development, especially during heavy metal stress.

Nectar Chemistry or Flower Morphology-What Is More Important for the Reproductive Success of Generalist Orchid Epipactis palustris in Natural and Anthropogenic Populations?

The aim of this study was to determine the level of reproductive success (RS) in natural and anthropogenic populations of generalist orchid Epipactis palustris and its dependence on flower structure and nectar composition, i.e., amino acids and sugars. We found that both pollinaria removal and female reproductive success were high and similar in all populations, despite differences in flower traits and nectar chemistry. Flower structures were weakly correlated with parameters of RS. Nectar traits were more important in shaping RS; although, we noted differentiated selection on nectar components in distinct populations. Individuals in natural populations produced nectar with a larger amount of sugars and amino acids. The sucrose to (fructose and glucose) ratio in natural populations was close to 1, while in anthropogenic ones, a clear domination of fructose and glucose was noted. Our results indicate that the flower traits and nectar composition of E. palustris reflect its generalist character and meet the requirements of a wide range of pollinators, differing according to body sizes, mouth apparatus, and dietary needs. Simultaneously, differentiation of nectar chemistry suggests a variation of pollinator assemblages in particular populations or domination of their some groups. To our knowledge, a comparison of nectar chemistry between natural and anthropogenic populations of orchids is reported for the first time in this paper.

Brassinolide Enhances the Level of Brassinosteroids, Protein, Pigments, and Monosaccharides in Wolffia arrhiza Treated with Brassinazole.

Brassinolide (BL) represents brassinosteroids (BRs)-a group of phytohormones that are essential for plant growth and development. Brassinazole (Brz) is as a synthetic inhibitor of BRs' biosynthesis. In the present study, the responses of Wolffia arrhiza to the treatment with BL, Brz, and the combination of BL with Brz were analyzed. The analysis of BRs and Brz was performed using LC-MS/MS. The photosynthetic pigments (chlorophylls, carotenes, and xanthophylls) levels were determined using HPLC, but protein and monosaccharides level using spectrophotometric methods. The obtained results indicated that BL and Brz influence W. arrhiza cultures in a concentration-dependent manner. The most stimulatory effects on the growth, level of BRs (BL, 24-epibrassinolide, 28-homobrassinolide, 28-norbrassinolide, catasterone, castasterone, 24-epicastasterone, typhasterol, and 6-deoxytyphasterol), and the content of pigments, protein, and monosaccharides, were observed in plants treated with 0.1 µM BL. Whereas the application of 1 µM and 10 µM Brz caused a significant decrease in duckweed weight and level of targeted compounds. Application of BL caused the mitigation of the Brz inhibitory effect and enhanced the BR level in duckweed treated with Brz. The level of BRs was reported for the first time in duckweed treated with BL and/or Brz.

TaCKX2.2 Genes Coordinate Expression of Other TaCKX Family Members, Regulate Phytohormone Content and Yield-Related Traits of Wheat.

TaCKX gene family members (GFMs) play essential roles in the regulation of cytokinin during wheat development and significantly influence yield-related traits. However, detailed function of most of them is not known. To characterize the role of TaCKX2.2 genes we silenced all homoeologous copies of both TaCKX2.2.1 and TaCKX2.2.2 by RNAi technology and observed the effect of silencing in 7 DAP spikes of T1 and T2 generations. The levels of gene silencing of these developmentally regulated genes were different in both generations, which variously determined particular phenotypes. High silencing of TaCKX2.2.2 in T2 was accompanied by slight down-regulation of TaCKX2.2.1 and strong up-regulation of TaCKX5 and TaCKX11, and expression of TaCKX1, TaCKX2.1, and TaCKX9 was comparable to the non-silenced control. Co-ordinated expression of TaCKX2.2.2 with other TaCKX GFMs influenced phytohormonal homeostasis. Contents of isoprenoid, active cytokinins, their conjugates, and auxin in seven DAP spikes of silenced T2 plants increased from 1.27 to 2.51 times. However, benzyladenine (BA) and abscisic acid (ABA) contents were significantly reduced and GA3 was not detected. We documented a significant role of TaCKX2.2.2 in the regulation of thousand grain weight (TGW), grain number, and chlorophyll content, and demonstrated the formation of a homeostatic feedback loop between the transcription of tested genes and phytohormones. We also discuss the mechanism of regulation of yield-related traits.

How Are the Flower Structure and Nectar Composition of the Generalistic Orchid Neottia ovata Adapted to a Wide Range of Pollinators?

Plant-pollinator interactions significantly influence reproductive success (RS) and drive the evolution of pollination syndromes. In the context of RS, mainly the role of flower morphology is touched. The importance of nectar properties is less studied, despite its significance in pollination effectiveness. Therefore, the aim of this study was to test selection on flower morphology and nectar chemistry in the generalistic orchid Neottia ovata. In 2019-2020, we measured three floral displays and six flower traits, pollinaria removal (PR), female reproductive success (FRS), and determined the soil properties. The sugars and amino acids (AAs) were analyzed using the HPLC method. Data were analyzed using multiple statistical methods (boxplots, ternary plot, one-way ANOVA, Kruskal-Wallis test, and PCA). Variation of flower structure and nectar chemistry and their weak correlation with RS confirms the generalistic character of N. ovata. In particular populations, different traits were under selection. PR was high and similar in all populations in both years, while FRS was lower and varied among populations. Nectar was dominated by glucose, fructose, and included 28 AAs (Ala and Glu have the highest content). Sugars and AAs influenced mainly FRS. Among soil parameters, carbon and carbon:nitrogen ratio seems to be the most important in shaping flower structure and nectar chemistry.

Intraspecific Variation in Nectar Chemistry and Its Implications for Insect Visitors: The Case of the Medicinal Plant, Polemonium Caeruleum L.

Floral nectar, being a primary reward for insect visitors, is a key factor in shaping plant-pollinator interactions. However, little is known about the variability in nectar traits, which could potentially affect pollinators and the reproduction of the species. We investigated intraspecific variation in nectar traits in 14 populations of a Red-listed plant, Polemonium caeruleum. Populations varied in terms of the proportion of self-compatible and self-incompatible individuals, and insect communities visiting flowers. Using HPLC, we determined the nectar sugar and amino acid (AA) composition and concentration. We also recorded some basic habitat parameters, which could influence nectar chemistry. In seven selected populations, we investigated the taxonomic composition of the insects visiting flowers. Our observations revealed significant intraspecific variability in nectar chemistry in P. caeruleum. Nectar production was male-biased, with male-phase flowers secreting sucrose- and AA-rich nectar. An analysis revealed that variability in P. caeruleum nectar may be slightly shaped by environmental factors. The studied nectar characters, especially sugars, had little effect on insects visiting flowers. We argue that variation in nectar traits in this generalist plant is a matter of random genetic drift or "adaptive wandering" rather than directional specialization and adaptation in the most effective and abundant group of pollinators.

Comprehensive Overview of the Brassinosteroid Biosynthesis Pathways: Substrates, Products, Inhibitors, and Connections.

Brassinosteroids (BRs) as a class of steroid plant hormones participate in the regulation of numerous developmental processes, including root and shoot growth, vascular differentiation, fertility, flowering, and seed germination, as well as in responding to environmental stresses. During four decades of research, the BR biosynthetic pathways have been well studied with forward- and reverse genetics approaches. The free BRs contain 27, 28, and 29 carbons within their skeletal structure: (1): 5α-cholestane or 26-nor-24α-methyl-5α-cholestane for C27-BRs; (2) 24α-methyl-5α-cholestane, 24ß-methyl-5α-cholestane or 24-methylene-5α-cholestane for C28-BRs; (3) 24α-ethyl-5α-cholestane, 24(Z)-ethylidene-5α-cholestane, 25-methyl-5α-campestane or 24-methylene-25-methyl-5α-cholestane for C29-BRs, as well as different kinds and orientations of oxygenated functions in A- and B-ring. These alkyl substituents are also common structural features of sterols. BRs are derived from sterols carrying the same side chain. The C27-BRs without substituent at C-24 are biosynthesized from cholesterol. The C28-BRs carrying either an α-methyl, ß-methyl, or methylene group are derived from campesterol, 24-epicampesterol or 24-methylenecholesterol, respectively. The C29-BRs with an α-ethyl group are produced from sitosterol. Furthermore, the C29 BRs carrying methylene at C-24 and an additional methyl group at C-25 are derived from 24-methylene-25-methylcholesterol. Generally, BRs are biosynthesized via cycloartenol and cycloartanol dependent pathways. Till now, more than 17 compounds were characterized as inhibitors of the BR biosynthesis. For nine of the inhibitors (e.g., brassinazole and YCZ-18) a specific target reaction within the BR biosynthetic pathway has been identified. Therefore, the review highlights comprehensively recent advances in our understanding of the BR biosynthesis, sterol precursors, and dependencies between the C27-C28 and C28-C29 pathways.

Author Correction: Diversity of nectar amino acids in the Fritillaria (Liliaceae) genus: ecological and evolutionary implications.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Diversity of nectar amino acids in the Fritillaria (Liliaceae) genus: ecological and evolutionary implications.

Nectar is considered to be a primary food reward for most pollinators. It mostly contains sugars, but also has amino acids. The significance of the concentration and composition of amino acids in nectar is often less understood than that of its volume, sugar concentration and composition. However, there is a trend towards a broader approach in ecological research, which helps to understand nectar properties in an ecological context. The genus Fritillaria, exhibiting great diversity in flower morphology, nectar composition, and dominant pollinators, allows for the possibility to study some of the above. We studied the concentration and composition of amino acids in the nectar of 38 Fritillaria species attracting different groups of pollen vectors (bees, flies, passerines, and hummingbirds). The flowers of fritillaries produced nectar with a varying composition and concentration of amino acids. These differences were mostly associated with the pollinator type. The nectar of passerine bird-pollinated species was rich in amino acids, whereas humming bird-pollinated produced low amino acid nectar. Contrary to previous reports nectar of the insect-pollinated species did not contain a higher amount of proline. Two non-protein amino acids, sarcosine and norvaline, were detected in the floral nectar for the first time.

Occurrence of brassinosteroids and influence of 24-epibrassinolide with brassinazole on their content in the leaves and roots of Hordeum vulgare L. cv. Golden Promise.

MAIN CONCLUSION: 24-epibrassinolide overcame the inhibitory effect of brassinazole on the barley growth and the content of brassinosteroids. The present work demonstrates the occurrence of mainly castasterone, brassinolide and cathasterone and lower amounts of 24-epibrassinolide, 24-epicastasterone, 28-homobrassinolide, typhasterol, 6-deoxocastasterone and 6-deoxotyphasterol in 14-day-old de-etiolated barley (Hordeum vulgare L. cv. Golden Promise). We also investigated the endogenous level of brassinosteroids (BRs) in barley seedlings treated with 24-epibrassinolide (EBL) and/or brassinazole (Brz). To our knowledge, this is the first report related to the occurrence of BRs and application of EBL and Brz in terms of the endogenous content of BRs in barley. Brz as a specific inhibitor of BR biosynthetic reactions decreased the level of BRs in the leaves. Application of EBL showed a weak promotive effect on the BR content in Brz-treated seedlings. Brz also inhibited growth of the seedlings; however, addition of EBL overcame the inhibition. The EBL applied alone at 0.01-1 µM increased the BR level in the leaves but at 10 µM lowered the BR content. In opposition to leaves, the Brz in the concentration range from 0.1 to 1 µM did not significantly affect the content of BRs in the roots. However, application of 10 µM Brz caused BRs to decrease, but treatment of EBL concentrations overcame the inhibitory effect of Brz.

Functional Diversity of Nectary Structure and Nectar Composition in the Genus Fritillaria (Liliaceae).

Fritillaria is a genus consisting of 130 to 140 species of bulbous plants, native to temperate regions of the northern hemisphere. Generally viewed as an insect pollinated genus with the exception of two North American species, Fritillaria gentneri and F. recurva, which are described as hummingbird-pollinated and the Asian species, F. imperialis, described as passerine-pollinated. These pollinator shifts are possibly the result of adaptive changes to the structure and morphology of the nectary, as well as a change in the nectar concentration and composition. A study was conducted in a target group of 56 Fritillaria species, based on the morphology of their nectaries and nectar composition to assess the significance of pollination mode as well as its predisposition for the evolution of bird pollination. All species studied had nectaries located at their tepal base and produced nectar, but their size, shape, color, and composition all varied. Most fritillaries had hexose-rich nectar, in easily accessible and unprotected nectaries. Scanning electron microscope (SEM) analysis revealed that the surface of the nectaries of most Fritillaria species was flat and clearly distinct from that of the surrounding tissues, which might be regarded as an adaptation for insect-pollination. Nectaries of F. imperialis were considerably larger and had dilute nectar without sucrose, which was produced profusely, thereby fulfilling the criteria characteristic of ornithophilous flowers. The copious nectar of presumed hummingbird-pollinated species was rather balanced and of medium sugar concentration. Their large lanceolate nectaries contrasted sharply with the tessellated background of their tepals. These characters might indicate a mixed pollination system that engages both birds and insects. Floral anatomy and microstructure and nectar composition for Fritillaria species in subgenera Korolkowia and Liliorhiza are studied for the first time.