Plants, Volume 10, Issue 11 (November 2021) – 322 articles

The present investigation was carried out to explore the biochemical changes and antioxidant variations, including non-enzymatic and enzymatic antioxidant variations, in the leaves of different varieties of date palm (Phoenix dactylifera L.) belonging to the early, mid-, and late-flowering categories in the United Arab Emirates. The changes in the protein and phenol concentration; the ascorbic acid, reduced glutathione, and α-tocopherol contents; and the activity of peroxidase and polyphenol oxidase were studied in the leaves during the preflowering, flowering, and postflowering stages of the date palms. Two varieties each from the early (Shaham, Khanezi), mid- (Barhee, Nabthasaif), and late- (Khasab, Fardh) flowering types were used in this study. The protein content in the leaves was higher in the early flowering varieties during the preflowering stage but lower in the other two varieties. The phenol content showed an opposite trend to the protein. There was significant variation in the ascorbic acid content and a reduction in glutathione and α-tocopherol between the leaves of different varieties. Similarly, the activity of the antioxidant enzyme ascorbate peroxidase in the leaves was higher during the preflowering stage in all varieties. The superoxide dismutase (SOD), polyphenol oxidase (PPO), and catalase (CAT) activity was highest in the Bharhee leaves for all the stages. The peroxidase activity (POD) was highest in the Fardh variety of date palm, whereas the Khanezi variety exhibited the lowest activity. This study can be used as a baseline for developing more protocols for understanding the possible roles of biochemicals, antioxidants, antioxidant enzymes, and their interactions in the regulation of flower development in different date palm varieties. Full article

Increasing soil salinity due to global warming severely restricts crop growth and yield. To select and recommend salt-tolerant cultivars, extensive genotypic screening and examination of plants’ morpho-physiological responses to salt stress are required. In this study, 18 prescreened maize hybrid cultivars were examined at the early growth stage under a hydroponic system using multivariate analysis to demonstrate the genotypic and phenotypic variations of the selected cultivars under salt stress. The seedlings of all maize cultivars were evaluated with two salt levels: control (without NaCl) and salt stress (12 dS m⁻¹ simulated with NaCl) for 28 d. A total of 18 morpho-physiological and ion accumulation traits were dissected using multivariate analysis, and salt tolerance index (STI) values of the examined traits were evaluated for grouping of cultivars into salt-tolerant and -sensitive groups. Salt stress significantly declined all measured traits except root–shoot ratio (RSR), while the cultivars responded differently. The cultivars were grouped into three clusters and the cultivars in Cluster-1 such as Prabhat, UniGreen NK41, Bisco 51, UniGreen UB100, Bharati 981 and Star Beej 7Star exhibited salt tolerance to a greater extent, accounting for higher STI in comparison to other cultivars grouped in Cluster-2 and Cluster-3. The high heritability (h²_bs, >60%) and genetic advance (GAM, >20%) were recorded in 13 measured traits, indicating considerable genetic variations present in these traits. Therefore, using multivariate analysis based on the measured traits, six hybrid maize cultivars were selected as salt-tolerant and some traits such as Total Fresh Weight (TFW), Total Dry Weight (TDW), Total Na⁺, Total K⁺ contents and K⁺–Na⁺ Ratio could be effectively used for the selection criteria evaluating salt-tolerant maize genotypes at the early seedling stage. Full article

Glucosinolates are well known as natural antimicrobials and anticarcinogenic agents. However, these compounds can lose their properties and transform into antinutrients, depending on processing conditions. In addition, the bitterness of some glucosinolate in rapeseed meal can affect the likability of the final product. Therefore, it is important to identify and determine each glucosinolate and its derived form, not just the total glucosinolate content, in order to evaluate the potential of the final rapeseed protein product. This study provides a comprehensive report of the types and quantities of glucosinolates and their derived forms (isothiocyanates) associated with different rapeseed processing conditions. Glucosinolates and isothiocyanates were determined by HPLC-DAD-qTOF. In our study, the enzymatic degradation of glucosinolates by myrosinase was the main factor affecting either glucosinolate or isothiocyanate content. Other factors such as pH seemed to influence the concentration and the presence of glucosinolates. In addition, process parameters, such as extraction time and separation technology, seemed to affect the amount and type of isothiocyanates in the final protein extracts. Overall, both determined intact glucosinolates and their derived forms of isothiocyanates can give different types of biological effects. More studies should be performed to evaluate the impact of glucosinolates and isothiocyanates on human health. Full article

Integrated Plant Nutrient System (IPNS) is practiced worldwide to maintain soil quality. Two field experiments were conducted in 2019 and 2020 in acidic and charland soils to assess the impact of different manures, viz., poultry manure (PM), vermicompost (VC), compost (OF), rice husk biochar (RHB), poultry manure biochar (PMB)-based IPNS, and dolomite over control on productivity and nitrogen use efficiency (NUE) of the Mustard-Boro-Transplanted Aman and Maize-Jute-Transplanted Aman cropping patterns, and on soil properties. The experiments were laid out in a randomized complete block design with four replications. The results showed that IPNS treatments significantly improved soil aggregate properties and total nitrogen in acidic soil, and bulk density in charland soil. In both years, IPNS treatments increased system productivity from 55.4 to 82.8% in acidic soil and from 43.3 to 115.4% in charland soil over that of control. IPNS and dolomite treatments increased nitrogen uptake from 35.5 to 105.7% over that of control and NUE in both soils in 2019 and 2020. PMB- and OF-based INPS treatments exhibited superior performances in both soils, and the impact was more prominent in 2020. Therefore, PMB- and OF-based IPNS can be recommended for maximizing system productivity and NUE with concurrent improvement of physicochemical properties of acidic and charland soils. Full article

Seed shape in species of the Cactaceae is described by comparison with geometric models. Three new groups of models are presented, two for symmetric seeds, and a third group for asymmetric seeds. The first two groups correspond, respectively, to superellipses and the combined equations of two semi-ellipses. The third group contains models derived from the representation of polar equations of Archimedean spirals that define the shape of asymmetric seeds in genera of different subfamilies. Some of the new models are geometric curves, while others are composed with a part resulting from the average silhouettes of seeds. The application of models to seed shape quantification permits the analysis of variation in seed populations, as well as the comparison of shape between species. The embryos of the Cactaceae are of the peripheral type, strongly curved and in contact with the inner surface of the seed coat. A relationship is found between seed elongation and the models, in which the genera with elongated seeds are represented by models with longer trajectories of the spiral. The analysis of seed shape opens new opportunities for taxonomy and allows quantification of seed shape in species of the Cactaceae. Full article

The incorporation of native plant species is central to restoration efforts, but this is often limited by both the availability of seeds and the relatively low viability and germination rates of commercially available seeds. Although pre-sowing treatments are commonly used to improve germination rates of seeds, the efficacy of these treatments is found to vary across species. In this study, we tested how four pre-sow treatments (physical scarification, acid scarification, cold stratification, and aerated hydropriming) affected the viability and seed germination rates of 12 commercially available plant species native to south Texas and commonly used in restoration efforts. Our results show that the viability of the seeds have a wide range, from 78% to 1.25%. Similarly, the total germination rate ranged from 62% to 0%. We found that pre-sowing treatments accelerated the germination rate in 9 of 12 plant species tested, but the effect varied by treatment. Collectively, our results identify various methods to achieve the best germination rates for native plants of south Texas, to help improve restoration efforts across the region. Full article

Previously, an efficient regeneration protocol was established and applied to regenerate plants from calli lines that could grow on eggplant leaf explants after a stepwise in vitro selection for tolerance to salt stress. Plants were regenerated from calli lines that could tolerate up to 120 mM NaCl. For further in vitro and in vivo evaluation, four plants with a higher number of leaves and longer roots were selected from the 32 plants tested in vitro. The aim of this study was to confirm the stability of salt tolerance in the progeny of these four mutants (‘R18’, ‘R19’, ‘R23’ and ‘R30’). After three years of in vivo culture, we evaluated the impact of NaCl stress on agronomic, physiological and biochemical parameters compared to the parental control (‘P’). The regenerated and control plants were assessed under in vitro and in vivo conditions and were subjected to 0, 40, 80 and 160 mM of NaCl. Our results show significant variation in salinity tolerance among regenerated and control plants, indicating the superiority of four regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) when compared to the parental line (‘P’). In vitro germination kinetics and young seedling growth divided the lines into a sensitive and a tolerant group. ‘P’ tolerate only moderate salt stress, up to 40 mM NaCl, while the tolerance level of ‘R18’, ‘R19’, ‘R23’ and ‘R30’ was up to 80 mM NaCl. The quantum yield of PSII (Φ_PSII) declined significantly in ‘P’ under salt stress. The photochemical quenching was reduced while nonphotochemical quenching rose in ‘P’ under salt stress. Interestingly, the regenerants (‘R18’, ‘R19’, ‘R23’ and ‘R30’) exhibited high apparent salt tolerance by maintaining quite stable Chl fluorescence parameters. Rising NaCl concentration led to a substantial increase in foliar proline, malondialdehyde and soluble carbohydrates accumulation in ‘P’. On the contrary, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ exhibited a decline in soluble carbohydrates and a significant enhancement in starch under salinity conditions. The water status reflected by midday leaf water potential (ψl) and leaf osmotic potential (ψπ) was significantly affected in ‘P’ and was maintained a stable level in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ under salt stress. The increase in foliar Na⁺ and Cl⁻ content was more accentuated in parental plants than in regenerated plants. The leaf K⁺, Ca²⁺ and Mg²⁺ content reduction was more aggravated under salt stress in ‘P’. Under increased salt concentration, ‘R18’, ‘R19’, ‘R23’ and ‘R30’ associate lower foliar Na⁺ content with a higher plant tolerance index (PTI), thus maintaining a normal growth, while foliar Na⁺ accumulation was more pronounced in ‘P’, revealing their failure in maintaining normal growth under salinity stress. ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an obvious salt tolerance by maintaining significantly high chlorophyll content. In ‘R18’, ‘R19’, ‘R23’ and ‘R30’, the enzyme scavenging machinery was more performant in the roots compared to the leaves. Salt stress led to a significant augmentation of catalase, ascorbate peroxidase and guaiacol peroxidase activities in the roots of ‘R18’, ‘R19’, ‘R23’ and ‘R30’. In contrast, enzyme activities were less enhanced in ‘P’, indicating lower efficiency to cope with oxidative stress than in ‘R18’, ‘R19’, ‘R23’ and ‘R30’. ACC deaminase activity was significantly higher in ‘R18’, ‘R19’, ‘R23’ and ‘R30’ than in ‘P’. The present study suggests that regenerated plants ‘R18’, ‘R19’, ‘R23’ and ‘R30’ showed an evident stability in tolerating salinity, which shows their potential to be adopted as interesting selected mutants, providing the desired salt tolerance trait in eggplant. Full article

This study is the first approach to in vitro asymbiotic germination of two species of Sicilian threatened terrestrial orchids, Anacamptis longicornu and Ophrys panormitana. Seeds were collected in the wild and cultured in two different media—Orchimax medium (OM) and Murashige and Skoog (MS)—and exposed to different photoperiods and temperatures to evaluate the best conditions for the specific stages of development. The germination of A. longicornu was very high on OM (95.5%) and lower on MS medium (21.4%), whereas O. panormitana germinated only on OM medium, with significantly lower percentages (12.0%), compared with A. longicornu. This difference is caused by variation in quality and quantity of nutrients used, primarily by nitrogen source. The results show that temperature and photoperiod widely affect seed germination and development. Although further investigations on asymbiotic and symbiotic germination are needed for the improvement of conservation of Mediterranean terrestrial orchids, our results contribute to the conservation of this group of plants. Full article

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants. Full article

The discovery of telomeric repeats in interstitial regions of plant chromosomes (ITRs) through molecular cytogenetic techniques was achieved several decades ago. However, the information is scattered and has not been critically evaluated from an evolutionary perspective. Based on the analysis of currently available data, it is shown that ITRs are widespread in major evolutionary lineages sampled. However, their presence has been detected in only 45.6% of the analysed families, 26.7% of the sampled genera, and in 23.8% of the studied species. The number of ITR sites greatly varies among congeneric species and higher taxonomic units, and range from one to 72 signals. ITR signals mostly occurs as homozygous loci in most species, however, odd numbers of ITR sites reflecting a hemizygous state have been reported in both gymnosperm and angiosperm groups. Overall, the presence of ITRs appears to be poor predictors of phylogenetic and taxonomic relatedness at most hierarchical levels. The presence of ITRs and the number of sites are not significantly associated to the number of chromosomes. The longitudinal distribution of ITR sites along the chromosome arms indicates that more than half of the ITR presences are between proximal and terminal locations (49.5%), followed by proximal (29.0%) and centromeric (21.5%) arm regions. Intraspecific variation concerning ITR site number, chromosomal locations, and the differential presence on homologous chromosome pairs has been reported in unrelated groups, even at the population level. This hypervariability and dynamism may have likely been overlooked in many lineages due to the very low sample sizes often used in cytogenetic studies. Full article

A study was carried out to evaluate the effectiveness of sugar beet extract (SBE) and glycine betaine (GB) in mitigating the adverse effects of drought stress on two maize cultivars. Seeds (caryopses) of two maize cultivars, Sadaf (drought-tolerant) and Sultan (drought-sensitive) were sown in plastic pots. Plants were subjected to different (100%, 75% and 60% field capacity (FC)) water regimes. Then, different levels of SBE (3% and 4%) and GB (3.65 and 3.84 g/L) were applied as a foliar spray after 30 days of water deficit stress. Drought stress significantly decreased plant growth and yield attributes, chlorophyll pigments, while it increased relative membrane permeability (RMP), levels of osmolytes (GB and proline), malondialdehyde (MDA), total phenolics and ascorbic acid as well as the activities of superoxide dismutase (SOD) and peroxidase (POD) enzymes in both maize cultivars. Exogenous application via foliar spray with SBR or GB improved plant growth and yield attributes, chlorophyll pigments, osmolyte concentration, total phenolics, ascorbic acid and the activities of reactive oxygen species (ROS) scavenging enzymes (SOD, POD and catalase; CAT), but reduced leaf RMP and MDA concentration. The results obtained in this study exhibit the role of foliar-applied biostimulants (natural and synthetic compounds) in enhancing the growth and yield of maize cultivars by upregulating the oxidative defense system and osmoprotectant accumulation under water deficit conditions. Full article

An efficient regeneration protocol was applied to regenerate shoots on salt stress-tolerant calli lines of aubergine (Solanum melongena). These NaCl-tolerant cell lines were obtained by two different methods. On the one hand, the developed callus tissue was transferred to a medium with a continuous salt content of 40, 80, 120, or 160 mM NaCl. On the other hand, the callus tissue was subjected to a stepwise increasing salinity to 160 mM NaCl every 30 days. With the second method, calli which could be selected were characterized by compact growth, a greenish color, and absence of necrotic zones. When grown on salt-free medium again, NaCl-tolerant calli showed a decline in relative growth rate and water content in comparison to the control line. This was more obvious in the 120 mM NaCl-tolerant callus. Lipid peroxidase activity increased in 40 and 80 mM NaCl-tolerant calli; yet did not increase further in 120 mM-tolerant callus. An increase in ascorbic acid content was observed in 80 and 120 mM NaCl-tolerant calli compared to the 40 mM NaCl-tolerant lines, in which ascorbic acid content was twice that of the control. All NaCl-tolerant lines showed significantly higher superoxide dismutase (SOD) (208–305–370 µmol min⁻¹ mg⁻¹ FW) and catalase (CAT) (136–211–238 µmol min⁻¹ mg⁻¹ FW) activities compared to control plants (231 and 126 µmol min⁻¹ mg⁻¹ FW). Plants were regenerated on the calli lines that could tolerate up to 120 mM NaCl. From the 32 plants tested in vitro, ten plants with a higher number of leaves and root length could be selected for further evaluation in the field. Their high salt tolerance was evident by their more elevated fresh and dry weight, their more increased relative water content, and a higher number and weight of fruits compared to the wild-type parental control. The presented work shows that somaclonal variation can be efficiently used to develop salt-tolerant mutants. Full article

The integumentary tissues of plant seeds protect the embryo (new sporophyte) forming in them from unfavorable external conditions; therefore, comprehensive knowledge about the structural and functional specificity of seed covers in various plants may be of both theoretical and practical interest. As a result of our study, additional data were obtained on the morphological and ultrastructural features of the formation of a multilayer skin of wheat (Triticum aestivum L.) kernel (caryopsis). The ultrastructure research analysis showed that differentiation of the pericarp and inner integument of the ovule leads to the formation of functionally different layers of the skin of mature wheat grain. Thus, the differentiation of exocarp and endocarp cells is accompanied by a significant thickening of the cell walls, which reliably protect the ovule from adverse external conditions. The cells of the two-layer inner integument of the ovule differentiate into cuticular and phenolic layers, which are critical for protecting daughter tissues from various pathogens. The epidermis of the nucellus turns into a layer of mucilage, which apparently helps to maintain the water balance of the seed. Morphological and ultrastructural data showed that the formation of the kernel’s skin occurs in coordination with the development of the embryo and endosperm up to the full maturity of the kernel. This is evidenced by the structure of the cytoplasm and nucleus, characteristic of metabolically active protoplasts of cells, which is observed in most integumentary layers at the late stages of maturation. This activity can also be confirmed by a significant increase in the thickness of the cell walls in the cells of two layers of the exocarp and in cross cells in comparison with the earlier stages. Based on these results, we came to the conclusion that the cells of a majority in the covering tissues of the wheat kernel during its ontogenesis are transformed into specialized layers of the skin by terminal differentiation. Full article

Calcifying macroalgae contribute significantly to the structure and function of tropical marine ecosystems. Their calcification and photosynthetic processes are not well understood despite their critical role in marine carbon cycles and high vulnerability to environmental changes. This study aims to provide a better understanding of the macroalgal calcification process, focusing on its relevance concerning seawater carbonate chemistry and its relationship to photosynthesis in three dominant calcified macroalgae in Thailand, Padina boryana, Halimeda macroloba and Halimeda opuntia. Morphological and microstructural attributes of the three macroalgae were analyzed and subsequently linked to their calcification rates and responses to inhibition of photosynthesis. In the first experiment, seawater pH, total alkalinity and total dissolved inorganic carbon were measured after incubation of the macroalgae in the light and after equilibration of the seawater with air. Estimations of carbon uptake into photosynthesis and calcification and carbon release into air were obtained thereafter. Our results provide evidence that calcification of the three calcified macroalgae is a potential source of CO₂, where calcification by H. opuntia and H. macroloba leads to a greater release of CO₂ per biomass weight than P. boryana. Nevertheless, this capacity is expected to vary on a diurnal basis, as the second experiment indicates that calcification is highly coupled to photosynthetic activity. Lower pH as a result of inhibited photosynthesis under darkness imposes more negative effects on H. opuntia and H. macroloba than on P. boryana, implying that they are more sensitive to acidification. These effects were worsened when photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, highlighting the significance of photosynthetic electron transport-dependent processes. Our findings suggest that estimations of the amount of carbon stored in the vegetated marine ecosystems should account for macroalgal calcification as a potential carbon source while considering diurnal variations in photosynthesis and seawater pH in a natural setting. Full article

We study here how soil bacterial communities of different ecosystems respond to disturbances caused by enrichments with monoterpenes that are common essential oil constituents. We used fenchone, 1,8-cineol and α-pinene, and soils from phrygana, a typical Mediterranean-type ecosystem where aromatic plants abound, and from another five ecosystem types, focusing on culturable bacteria. Patterns of response were common to all ecosystems, but responses themselves were not always as pronounced in phrygana as in the other ecosystems, suggesting that these enrichments are less of a disturbance there. More specifically, soil respiration and abundance of the bacterial communities increased, becoming from below two up to 16 times as high as in control soils (for both attributes) and remained at high levels as long as these compounds were present. Bacteria that can utilize these three compounds as substrates of growth became dominant members of the bacterial communities in the enriched soils. All changes were readily reversible once monoterpene addition stopped. Bacteria with the ability to utilize these monoterpenes as carbon sources were found in soils from all ecosystems, 15 strains in total, suggesting a rather universal presence; of these, six could also utilize the organic pollutants toluene or p-xylene. These results suggest also potential novel applications of monoterpenes in combating soil pollution. Full article

Roots are the most important plant organ for absorbing essential elements, such as water and nutrients for living. To develop new climate-resilient soybean cultivars, it is essential to know the variation in root morphological traits (RMT) among diverse soybean for selecting superior root attribute genotypes. However, information on root morphological characteristics is poorly understood due to difficulty in root data collection and visualization. Thus, to overcome this problem in root research, we used a 2-dimensional (2D) root image in identifying RMT among diverse soybeans in this research. We assessed RMT in the vegetative growth stage (V2) of 372 soybean cultivars propagated in polyvinyl chloride pipes. The phenotypic investigation revealed significant variability among the 372 soybean cultivars for RMT. In particular, RMT such as the average diameter (AD), surface area (SA), link average length (LAL), and link average diameter (LAD) showed significant variability. On the contrary RMT, as with total length (TL) and link average branching angle (LABA), did not show differences. Furthermore, in the distribution analysis, normal distribution was observed for all RMT; at the same time, difference was observed in the distribution curve depending on individual RMT. Thus, based on overall RMT analysis values, the top 5% and bottom 5% ranked genotypes were selected. Furthermore, genotypes that showed most consistent for overall RMT have ranked accordingly. This ultimately helps to identify four genotypes (IT 16538, IT 199127, IT 165432, IT 165282) ranked in the highest 5%, whereas nine genotypes (IT 23305, IT 208266, IT 165208, IT 156289, IT 165405, IT 165019, IT 165839, IT 203565, IT 181034) ranked in the lowest 5% for RMT. Moreover, principal component analysis clustered cultivar 2, cultivar 160, and cultivar 274 into one group with high RMT values, and cultivar 335, cultivar 40, and cultivar 249 with low RMT values. The RMT correlation results revealed significantly positive TL and AD correlations with SA (r = 0.96) and LAD (r = 0.85), respectively. However, negative correlations (r = −0.43) were observed between TL and AD. Similarly, AD showed a negative correlation (r = −0.22) with SA. Thus, this result suggests that TL is a more vital factor than AD for determining SA compositions. Full article

Several genera formerly contained within the genus Sorbus L. sensu lato have been proposed as separate taxa, including Aria, Chamaemespilus and Torminalis. However, molecular evidence for such distinctions are rather scarce. We assembled the complete chloroplast genome of Sorbus aucuparia, another representative of Sorbus s.s., and performed detailed comparisons with the available genomes of Aria edulis, Chamaemespilus alpina and Torminalis glaberrima. Additionally, using 110 complete chloroplast genomes of the Maleae representatives, we constructed the phylogenetic tree of the tribe using Maximum Likelihood methods. The chloroplast genome of S. aucuparia was found to be similar to other species within Maleae. The phylogenetic tree of the Maleae tribe indicated that A. edulis, C. alpina and T. glaberrima formed a concise group belonging to a different clade (related to Malus) than the one including Sorbus s.s. (related to Pyrus). However, Aria and Chamaemespilus appeared to be more closely related to each other than to Torminalis. Our results provide additional support for considering Aria, Chamaemespilus and Torminalis as separate genera different from Sorbus s.s. Full article

Changes in stomatal conductance and density allow plants to acclimate to changing environmental conditions. In the present paper, the influence of atmospheric CO₂ concentration and light intensity on stomata were investigated for two barley genotypes—Barke and Bojos, differing in their sensitivity to oxidative stress and phenolic acid profiles. A novel approach for stomatal density analysis was used—a pair of convolution neural networks were developed to automatically identify and count stomata on epidermal micrographs. Stomatal density in barley was influenced by genotype, as well as by light and CO₂ conditions. Low CO₂ conditions resulted in increased stomatal density, although differences between ambient and elevated CO₂ were not significant. High light intensity increased stomatal density compared to low light intensity in both barley varieties and all CO₂ treatments. Changes in stomatal conductance were also measured alongside the accumulation of pentoses, hexoses, disaccharides, and abscisic acid detected by liquid chromatography coupled with mass spectrometry. High light increased the accumulation of all sugars and reduced abscisic acid levels. Abscisic acid was influenced by all factors—light, CO₂, and genotype—in combination. Differences were discovered between the two barley varieties: oxidative stress sensitive Barke demonstrated higher stomatal density, but lower conductance and better water use efficiency (WUE) than oxidative stress resistant Bojos at saturating light intensity. Barke also showed greater variability between treatments in measurements of stomatal density, sugar accumulation, and abscisic levels, implying that it may be more responsive to environmental drivers influencing water relations in the plant. Full article

Fig (Ficus carica L.) leaves are produced each year and often disposed, resulting in a waste of resources. Fig waste leaves are rich in flavonoids, which have strong antioxidant activity; however, the variety and chemical structure of antioxidants in fig leaves have not been reported in detail. To take full advantage of fig waste leaves, antioxidant capacity of different extracts (petroleum ether, ethyl acetate, and water) was evaluated by 1, 1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), and ferric-ion-reducing antioxidant power (FRAP) methods. The results showed that flavonoids in ethyl acetate extraction had the highest content (83.92 ± 0.01 mg/g), maximum DPPH scavenging activity (IC₅₀ 0.54 mg/mL), highest ABTS scavenging rate (80.28%), and FRAP (3.46 mmol/g). Furthermore, an HPLC-QTOF-MS/MS-DPPH method was developed to identify 11 flavonoids in fig waste leaves. This rapid and efficient method can not only be used for screening the antioxidant components in fig waste leaves, but also can be combined with mass spectrometry to identify the compounds with antioxidant capacity. There are three flavonoids with significant antioxidant capacity, which are 3-O-(rhamnopyranosyl-glucopyranosyl)-7-O-(glucopyranosyl)-quercetin, isoschaftoside, and rutin. The results confirmed that fig waste leaves contain a variety of antioxidant components, which contributed to increase the value of fig waste leaves as antioxidants. Full article

Phenolic compounds isolated from plant biomass consist of bioactive components showing a wide range of benefits for humans, including antioxidant, antimicrobial or anti-inflammatory effects. This paper presents the potential value of black alder (Alnus glutinosa (L.) Gaertn. (Betulaceae)) bark for the production of biologically active substances, despite its current use as a low value fuel source. Most of the extraction methods employ neat organic solvents to obtain extracts with a high antioxidant potential from biomass. The aim of this work is to show the advantages and disadvantages of the extraction process by taking into account the principles of ‘green chemistry’ and replacing the organic solvents with ‘green’ solvent water. Using the advantages of accelerated solvent extraction (ASE), it has been shown that the use of deionized water has the prospect of replacing organic solvents. In the case of the one-step water extraction, the total polyphenol content (TPC) varies from 0.55 to 0.62 Gallic acid equivalent (GAE) g/g in the extracts, depending on the temperature, whereas with the result of the sequential extraction with the organic solvents, the TPC content of the 40% (v:v) ethanol extracts ranges from 0.39 to 0.61 GAE g/g, depending on the temperature. The influence of the total polyphenol content and the total proanthocyanidin content on the antioxidant activity is shown. The antioxidant activity (IC₅₀, mg/L) of the extracts obtained with the organic solvents in the (2,2-diphenyl-1-picrylhydrazyl) DPPH^• test varies from 4.05 to 9.58, depending on the temperature in the range of 70–150 °C, respectively, while the results obtained with the deionized water showed promising results in the range of 6.33–7.36 in the temperature range of 70–150 °C, respectively. The extraction with the deionized water showed that approximately 90% of the substances in the extracts obtained with the organic solvents by sequential extraction are possible to obtain as deionized water extracts. Full article

Algae and fungi share a rich history in the fields of basic and applied natural science. In biotechnology, in particular, algae and fungi are of paramount importance, due to the production and development of valuable compounds, such as pharmaceuticals, enzymes, and biofuels. They are also used in waste fermentation, biocontrol of pathogens, and food processing and improvement, among other fields. Although a substantial number of different microorganisms are utilized for these purposes, there lies tremendous potential in uncharacterized microbial species. For this reason, biodiversity hotspots offer a wealth of potential in the discovery of new products and processing strategies based on these microorganisms. This review presents an overview of the use of algae and fungi in pre-Hispanic times/modern-day Mexico for the benefits of mankind. One of our objectives is to raise awareness about the potential of developing research projects for identification and biotechnological utilization of algae and fungi in a megadiverse country, such as Mexico. Full article

The composition of free volatile compounds of essential oils (EO) and hydrosols (Hy) from four different localities of the species Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fisch. were analyzed by gas chromatography coupled with mass spectrometry. In the EOs, the most abundant compounds identified were hexahydrofarnesyl acetone (23.34–52.56%), hexadecanoic acid (palmitic acid, 26.71–58.91%) and octadecanol acetate (0–6.24%). The hydrosols were characterized by high abundance of methyl eugenol (23.35–57.93%), trans-p-mentha-1(7),8-dien-2-ol (5.24–7.69%) and thymol (3.48–9.45%). Glandular trichomes were analyzed using SEM (Scanning Electron Microscopy), as they are the sites of synthesis of free volatile compounds. We have detected glandular trichomes, consisting of a one stalk cell and two elliptically shaped head cells, and non-glandular (unbranched, bi-cellular to multicellular) trichomes on stems, leaves and the sepals. Data for volatile compounds from EOs and hydrosols were analyzed using Principal Component Analyses (PCA) to demonstrate variations in the composition of the volatile compounds identified. Isolated samples of EO and hydrosols were analyzed for their antioxidant activity using two methods, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (Oxygen Radical Absorbance Capacity). The essential oils showed higher antioxidant activity than the hydrosols in ORAC method, but lower activity by the DPPH method. The isolates were also tested for their antiproliferative activity on different types of cancer cells and also on two lines of healthy cells, and the results showed that the extracts were not toxic to the cell lines tested. Total polyphenols, total tannins, total flavonoids and total phenolic acids were also analyzed and determined spectrophotometrically. The free volatile compounds of Veronica austriaca ssp. jacquinii can be considered as a safe natural product. Full article

Production of functional food with high levels of selenium (Se) and iodine (I) obtained via plant biofortification shows significant difficulties due to the complex interaction between the two elements. Taking into account the known beneficial effect of silicon (Si) on plant growth and development, single and joint foliar biofortification of chervil plants with potassium iodide (150 mg L⁻¹) and sodium selenate (10 mg L⁻¹) was carried out in a pot experiment with and without Si nanoparticles foliar supplementation. Compared to control plants, nano-Si (14 mg L⁻¹) increased shoot biomass in all treatments: by 4.8 times with Si; by 2.8 times with I + Si; by 5.6 times with Se + Si; by 4.0 times with I + Se + Si. The correspondent increases in root biomass were 4.5, 8.7, 13.3 and 10.0 times, respectively. The growth stimulation effect of Se, I and I + Se treatments resulted in a 2.7, 3.5 and 3.6 times increase for chervil shoots and 1.6, 3.1 and 8.6 times for roots, respectively. Nano-Si improved I biofortification levels by twice, while I and Se enhanced the plant content of each other. All treatments decreased nitrate levels, compared to control, and increased the photopigment accumulation. Improvement of total antioxidant activity and phenolic content was recorded only under the joint application of Se + I + Si. Foliar nano-Si treatment affected other element content in plants: decreased Na⁺ accumulation in single and joint supplementation with Se and I, restored Fe, Mn and Cr amount compared to the decreased levels recorded in separately Se and I fortified plants and promoted Al accumulation both with or without Se and I biofortification. The results of this research suggest high prospects of foliar nano-Si supply for enhancing both growth and joint I/Se biofortification of chervil. Full article

Barley production is essential in Egypt. In the present study, 15 different six-rowed Egyptian barley cultivars were studied. To differentiate between the different cultivars under study in terms of morphological characteristics and ISSR, molecular characterization reactions were carried out. Moreover, four cultivars (Giza 123, Giza 126, Giza 136, and Giza 138) were selected for further studies using scanning electron microscopy (SEM). Computational analysis of the DNA barcoding sequences of the two plastid markers rbcL and matK was executed, and the results were deposited in the NCBI database. The morphological traits showed low statistical significance among the different cultivars under study via the data collected from two seasons, suggesting that the mean field performance of these Egyptian cultivars may be equal under these conditions. The results showed that the phylogenetic tree was divided into four groups, one of which contained the most closely related genotypes in the genetic distance, including Giza 124, Giza 130, Giza 138, Giza 136, and Giza 137, which converge in the indicative uses of farmers. The seed coat of the studied cultivars was “rugose”. The elevation folding of the rugose pattern ranged from 11 ± 1.73 µm (Giza 126) to 14.67 ± 2.43 µm (Giza 123), suggesting variation in seed quality and its uses in feed and the food industry. According to the similarity matrix of ISSR analysis, the highest similarity value (93%) was recorded between Giza 133 and Giza 132, as well as between Giza 2000 and Giza 126. On the other hand, the lowest similarity value (80%) was recorded between Giza 130 and (Giza 133 and Giza 132), indicating that these cultivars were distantly related. Polymorphism information content (PIC) ranged from 0.26 for the primer ISSR UBC 835 to 0.37 for the primers ISSR UBC 814 and ISSR UBC 840. The current study showed that the matK gene is more mutable than the rbcL gene among the tested cultivars. Full article

Aspidosperma pyrifolium is used in traditional medicine to treat inflammatory disorders. The aim of the study was to perform phytochemical characterization and evaluate the anti-inflammatory, anti-nociceptive and acute toxicity effects of the total alkaloid fraction (TAF-Ap) from stem barks. Two monoterpenic indole alkaloids were isolated by high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) and the structural elucidation was performed using 1D and 2D NMR analysis. As for toxicity, no animals died at 50 mg kg⁻¹ and this concentration presented mild sedation and forced breathing within the first 24 h. The lethal dose capable of killing 50% of the animals (LD₅₀) was estimated to be 160 mg kg⁻¹. In the pharmacological tests, the models used were 1% carrageenan-induced paw edema and peritonitis, 1% formalin-induced nociception and 1% acetic acid-induced abdominal writhing in Swiss mice. The study made it possible to isolate 15-methoxyaspidospermine and 15-methoxypyrifolidine, corroborating the results of pharmacological assays, which showed anti-inflammatory and analgesic potential, especially at 30 mg kg⁻¹ (p < 0.001). Thus, the species was shown to be a promising source of active substances, with special attention paid to its toxicological potential. Full article

Prickly rose (Rosaacicularis Lindl.) is the most distributed rose species in the Northern Hemisphere, used by indigenous people for various food purposes. The lack of detailed information about the chemical composition of R. acicularis has led us to study the phytochemical composition and metabolic profile of prickly rose extracts using chromatographic techniques. Many groups of phenolic and non-phenolic compounds were quantified in the leaves, flowers, roots and fruits of R. acicularis. Phenolic compounds were the dominant phytochemicals in the aerial parts and roots of R. acicularis. A precise study by high-performance liquid chromatography with photodiode array detection and electrospray ionization triple quadrupole mass spectrometric detection showed the presence of 123 compounds, among which ellagic acid derivatives, ellagitannins, gallotannins, catechins, catechin oligomers, hydroxycinnamates and flavonoid glycosides of kaempferol, quercetin and dihydroquercetin were all identified for the first time. The most abundant phenolic compounds were ellagitannins and flavonoid glycosides, with a maximal content of 70.04 mg/g in leaves and 66.72 mg/g in flowers, respectively, indicating the great ability of R. acicularis organs to accumulate phenolic compounds. By applying a standardized static, simulated gastrointestinal digestion method, we found the inhibitory potential of the leaf extract against digestive α-amylases. A pancreatic α-amylase activity-inhibiting assay coupled with HPLC microfractionation demonstrated high inhibition of enzyme activity by ellagitannin rugosin D, which was later confirmed by a microplate reaction with mammalian α-amylases and the simulated digestion method. This study clearly demonstrates that R. acicularis leaf extract and its main component, ellagitannin rugosin D, strongly inhibit digestive α-amylase, and may be a prospective antidiabetic agent. Full article

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity. Full article

Bryophytes are poikilohydric organisms that play a key role in ecosystems, while some of them are also resistant to drought and environmental disturbances but present a slow growth rate. Moss culture in the laboratory can be a very useful tool for ecological restoration or the development of urban green spaces (roof and wall) in the Mediterranean region. Therefore, we aim to: (i) determine the optimal culture conditions for the growth of four moss species present in the Mediterranean climate, such as Bryum argenteum, Hypnum cupressiforme, Tortella nitida, and Tortella squarrosa; (ii) study the optimal growth conditions of the invasive moss Campylopus introflexus to find out if it can be a threat to native species. Photoperiod does not seem to cause any recognisable pattern in moss growth. However, temperature produces more linear but slower growth at 15 °C than at 20 and 25 °C. In addition, the lower temperature produced faster maximum cover values within 5–8 weeks, with at least 60% of the culture area covered. The study concludes that the culture of moss artificially in the organic gardening substrate without fertilisers is feasible and could be of great help for further use in environmental projects to restore degraded ecosystems or to facilitate urban green spaces in the Mediterranean area. Moreover, this study concludes that C. introflexus could successfully occupy the niche of other native moss species, especially in degraded areas, in a future global change scenario. Full article

Dermatophyte infections represent a significant public health concern, with an alarming negative impact caused by unsuccessful therapeutic regimens. Natural products have been highlighted as a promising alternative, due to their long-standing traditional use and increasing scientific recognition. In this study, honokiol and magnolol, the main bioactives from Magnolia spp. bark, were investigated for their antidermatophytic activity. The antifungal screening was performed using dermatophyte standard strains and clinical isolates. The minimal inhibitory concentration (MIC) and the minimal fungicidal concentration (MFC) were determined in accordance with EUCAST-AFST guidelines, with minor modifications. The effects on ergosterol biosynthesis were assessed in Trichophyton rubrum cells by HPLC-DAD. Putative interactions with terbinafine against T. rubrum were evaluated by the checkerboard method. Their impact on cells’ viability and pro-inflammatory cytokines (IL-1β, IL-8 and TNF-α) was shown using an ex vivo human neutrophils model. Honokiol and magnolol were highly active against tested dermatophytes, with MIC and MFC values of 8 and 16 mg/L, respectively. The mechanism of action involved the inhibition of ergosterol biosynthesis, with accumulation of squalene in T. rubrum cells. Synergy was assessed for binary mixtures of magnolol with terbinafine (FICI = 0.50), while honokiol-terbinafine combinations displayed only additive effects (FICI = 0.56). In addition, magnolol displayed inhibitory effects towards IL-1β, IL-8 and TNF-α released from lipopolysaccharide (LPS)-stimulated human neutrophils, while honokiol only decreased IL-1β secretion, compared to the untreated control. Overall, honokiol and magnolol acted as fungicidal agents against dermatophytes, with impairment of ergosterol biosynthesis. Full article

The increasing popularity of pomegranate (Punica granatum L.), driven by the awareness of its nutraceutical properties and excellent environmental adaptability, is promoting a global expansion of its production area. This investigation reports the variability in the weight, moisture, pH, total soluble solids, carbohydrates, organic acids, phenolic compounds, fatty acids, antioxidant activities, and element composition of different fruit parts (juices, peels, and kernels) from four (Ako, Emek, Kamel, and Wonderful One) of the most widely cultivated Israeli pomegranate varieties in Salento (South Italy). To the best of our knowledge, this is the first systematic characterization of different fruit parts from pomegranate cultivars grown simultaneously in the same orchard and subjected to identical agronomic and environmental conditions. Significant genotype-dependent variability was observed for many of the investigated parameters, though without any correlation among fruit parts. The levels of phenols, flavonoids, anthocyanins, and ascorbic and dehydroascorbic acids of all samples were higher than the literature-reported data, as was the antioxidant activity. This is likely due to positive interactions among genotypes, the environment, and good agricultural practices. This study also confirms that pomegranate kernels and peels are, respectively, rich sources of punicic acid and phenols together, with several other bioactive molecules. However, the variability in their levels emphasizes the need for further research to better exploit their agro-industrial potential and thereby increase juice-production chain sustainability. This study will help to assist breeders and growers to respond to consumer and industrial preferences and encourage the development of biorefinery strategies for the utilization of pomegranate by-products as nutraceuticals or value-added ingredients for custom-tailored supplemented foods. Full article