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The aim of this study was to determine the enzymatic profile of esterases and peroxidases during early stages of somatic embryogenesis of Fritillaria meleagris L. Somatic embryogenesis was induced using the leaf base as explant on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Zymography showed the presence of different moieties, six isoforms of esterases and peroxidases, during morphogenesis as compared to control explants. One isoform of esterases was detected only during the process of somatic embryogenesis, and one isoform was detected in control explants. Analysis of esterases with 1-naphthyl butyrate proved that esterases, which participate in somatic embryogenesis of F. meleagris, belong to the family of aryl esterases. For the first time it was proved that five isoforms of esterases, which are involved in morphogenesis of F. meleagris, belong to the family of aryl esterases, while two isoforms are carboxyl esterases. One isoform of carboxyl esterases was visible in control explants. This is also the first description of peroxidases during the morphogenetic process, and of the difference between aryl and carboxyl esterases. More isoforms of esterases during morphogenesis as compared to control explants are probably responsible for some early physiological process during somatic embryogenesis of F. meleagris. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON173015 and Grant no. OI 172048]

Over 20% of all protein domains are currently annotated as “domains of unknown function” or DUFs. In a recently identified Centaurium erythraea arabinogalactan peptide, CeAGP3 (AGN92423), a conserved DUF1070 domain was found. Since identifying functions for DUFs is important in systems biology, we have analyzed the distribution and structure of DUF1070 domain (pfam06376) using a set of bioinformatics tools. There are 271 publically available DUF1070 members from 25 diverse families of vascular plants, and most are short sequences (50-100 aa). The N-terminal signal peptide (Nsp) was found in almost all complete sequences. In 233 sequences, at least two noncontiguous prolines were found as clustered dipeptides predicted to be hydroxylated and glycosylated with type II arabino-3,6-galactans, thus representing AG-II glycomodules. In addition, 35 sequences contained a region rich in basic residues (basic linker, BL). The N-terminal part of the DUF1070 domain is comprised of (part of) AG-II and/or BL, while the highly conserved C-terminus is a region of 26 aa, termed SH26. In 212 sequences, SH26 was a typical glycosylphosphatidylinositol lipid anchor signal peptide (GPIsp), but in 83 cases GPIsp was not predicted due to software constraints. In sequences where both Nsp and GPIsp were predicted, the length of mature peptides could be calculated, and it was 10-16 aa. Our analysis suggests that DUF1070 members are arabinogalactan (AG) peptides, of which the majority are GPI-anchored. DUF1070 is the only conserved domain found in classical arabinogalactan proteins and AG peptides. The SH26 region can be used for mining and annotation of AG peptides. [Projekat Ministarstva nauke Republike Srbije, br. ТR31019]

Common centaury ( Centaurium eryhtraea Rafn) is a medicinal plant species with vigorous morphogenic potential in vitro . The process of spontaneous shoot regeneration in a solid root culture is characteristic for this plant species. In this context, the aim of this work was to investigate the dynamic changes of endogenous phytohormones and carbohydrates content in root explants at different time points (0, 2, 4, 7, 14, 21, 28, and 60 days) during spontaneous centaury morphogenesis in vitro . Detailed analysis of cytokinins (CKs) showed that trans -zeatin ( tZ ) was the major bioactive CK at all time points. The corresponding riboside, t Z9R, was also determined in the majority of the identified transport forms, at all time-points. Further analysis of endogenous auxin revealed a significant increase in endogenous indole-3-acetic acid (IAA) after 21 days, when a huge jump in the ratio of IAA/bioactive CKs was also observed. The maximum total soluble sugar content was measured after 14 days, while a significant decrease was determined after 21 days, when the first regenerated adventitious shoots appeared. This undoubtedly indicates an increased energy requirement prior to the actual regeneration of the shoots. The obtained results indicate that the period from day 14 to day 21 involves the most dramatic disturbances in endogenous bioactive CKs, IAA and carbohydrate balance, which are very important and valuable factors for the onset of shoot regeneration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Trifunović-Momčilov, Motyka, Marković, Milovančević, Filipović, Dobrev and Subotić.)

Background: Carrot is an important vegetable crop grown worldwide. The major economic problem in carrot cultivation is yellow disease caused by Bactericera trigonica, which induces biotic stress and has the greatest impact on crop productivity. Comprehensive studies on the mechanism of carrot defense response to biotic stress caused by B. trigonica infestation have yet to be conducted., Methods: The changes in photosynthetic pigments, proline, TPC, H 2 O 2 and MDA content, DPPH radical scavenging ability, and antioxidant enzyme activity of SOD, CAT, and POX in carrot leaves in response to insect sex (female and male), rapid response (during the first six hours), and long-term response to B. trigonica infestation were evaluated., Results: The results of our study strongly suggest that B. trigonica infestation causes significant changes in primary and secondary metabolism and oxidative status of carrot leaves. Photosynthetic pigment content, TPC, and DPPH and CAT activities were significantly reduced in carrot leaves in response to insect infestation. On the other hand, proline, H 2 O 2 content, and the activity of the antioxidant enzymes superoxide dismutase and peroxidase were increased in carrot leaves after B. trigonica infestation. The results indicate that B. trigonica attenuates and delays the oxidative stress responses of carrot, allowing long-term feeding without visible changes in the plant. Carrot responded to long-term B. trigonica infestation with an increase in SOD and POX activity, suggesting that these enzymes may play a key role in plant defense mechanisms., Conclusions: This is the first comprehensive study strongly suggesting that B. trigonica infestation causes significant changes in primary and secondary metabolism and an attenuated ROS defense response in carrot leaves that enables long-term insect feeding. The information provides new insights into the mechanisms of carrot protection against B. trigonica infestation., (© 2024. The Author(s).)

In this study, the effects of foliar applied methyl jasmonate (MeJA) on drought-stressed Impatiens walleriana growth and leaf physiology parameters: stomatal conductance, chlorophyll, flavonoid, anthocyanin, and nitrogen balance index (NBI), were evaluated. These parameters could serve as indicators of drought tolerance of I. walleriana , a popular horticultural plant worldwide that is very sensitive to drought. The experiment included four treatments: control, drought-stressed plants sprayed with distilled water, drought-stressed plants sprayed with 5 µM MeJA, and drought-stressed plants sprayed with 50 µM MeJA. Foliar spraying with MeJA was performed twice: seven days before and on the day of drought induction. The stressed plant groups were non-irrigated to achieve soil water contents (SWC) of 15 and 5%, while control plants were well-watered throughout the experiment (35-37% SWC). The results of this study showed that drought significantly reduced I. walleriana fresh and dry shoot weight, as well as total leaf area, but did not impact on dry matter content. The foliar application of MeJA improved growth parameters of I. walleriana , depending on the elicitor concentration and drought intensity. Stomatal conductance was slightly reduced at 5% SWC, and foliar applied MeJA at both concentrations. The flavonoid index was slightly reduced at 15 and 5% SWC when 50 µM MeJA was foliar applied, while there were no observed changes in the anthocyanin index in any treatments. The foliar application of 50 µM MeJA increased the chlorophyll index and NBI of I. walleriana at 5% SWC, indicating a contribution of the elicitor to plant drought tolerance at the physiological level.

The rhizosphere represents a center of complex and dynamic interactions between plants and microbes, resulting in various positive effects on plant growth and development. However, less is known about the effects of indole-3-acetic acid (IAA) on aquatic plants. In this study, we report the characterization of four Pseudomonas strains isolated from the rhizosphere of the common duckweed ( Lemna minor ) with IAA-degradation and -utilization ability. Our results confirm previous reports on the negative effect of IAA on aquatic plants, contrary to the effect on terrestrial plants. P. putida A3-104/5 demonstrated particularly beneficial traits, as it exhibited not only IAA-degrading and -producing activity but also a positive effect on the doubling time of duckweeds in the presence of IAA, positive chemotaxis in the presence of IAA, increased tolerance to oxidative stress in the presence of IAA and increased biofilm formation related to IAA. Similarly, P. gessardii C31-106/3 significantly shortened the doubling time of duckweeds in the presence of IAA, while having a neutral effect in the absence of IAA. These traits are important in the context of plant-bacteria interactions and highlight the role of IAA as a common metabolite in these interactions, especially in aquatic environments where plants are facing unique challenges compared to their terrestrial counterparts. We conclude that IAA-degrading and -producing strains presented in this study might regulate IAA effects on aquatic plants and confer evolutionary benefits under adverse conditions (e.g., under oxidative stress, excess of IAA or nutrient scarcity).

Drought stress affects plant growth and development through several mechanisms, including the induction of oxidative stress. To cope with drought, plants have drought tolerance mechanisms at the physiological, biochemical, and molecular levels. In this study, the effects of foliar application of distilled water and methyl jasmonate (MeJA) (5 and 50 µM) on the physiological, biochemical, and molecular responses of Impatiens walleriana during two drought regimes (15 and 5% soil water content, SWC) were investigated. The results showed that plant response depended on the concentration of the elicitor and the stress intensity. The highest chlorophyll and carotenoid contents were observed at 5% SWC in plants pre-treated with 50 µM MeJA, while the MeJA did not have a significant effect on the chlorophyll a/b ratio in drought-stressed plants. Drought-induced formation of hydrogen peroxide and malondialdehyde in plants sprayed with distilled water was significantly reduced in plant leaves pretreated with MeJA. The lower total polyphenol content and antioxidant activity of secondary metabolites in MeJA-pretreated plants were observed. The foliar application of MeJA affected the proline content and antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase) in plants that suffered from drought. The expression of abscisic acid (ABA) metabolic genes ( IwNCED4 , IwAAO2 , and IwABA8ox3 ) was the most affected in plants sprayed with 50 µM MeJA, while of the four analyzed aquaporin genes ( IwPIP1 ; 4 , IwPIP2 ; 2 , IwPIP2 ; 7 , and IwTIP4 ; 1 ), the expression of IwPIP1 ; 4 and IwPIP2 ; 7 was strongly induced in drought-stressed plants pre-treated with 50 µM MeJA. The study's findings demonstrated the significance of MeJA in regulating the gene expression of the ABA metabolic pathway and aquaporins, as well as the considerable alterations in oxidative stress responses of drought-stressed I. walleriana foliar sprayed with MeJA. The results improved our understanding of this horticulture plant's stress physiology and the field of plant hormones' interaction network in general.

Soil salinity is one of the most common abiotic stressors that affects plant growth and development. The aim of this work was to investigate the influence of sodium nitroprusside (SNP), a donor of nitric oxide (NO), on the physiological response of common centaury ( Centaurium erythraea ) shoots grown under stress conditions caused by sodium chloride (NaCl) in vitro. Centaury shoots were first grown on nutrient medium containing different SNP concentrations (50, 100 and 250 μM) during the pretreatment phase. After three weeks, the shoots were transferred to nutrient media supplemented with NaCl (150 mM) and/or SNP (50, 100 or 250 μM) for one week. The results showed that salinity decreased photosynthetic pigments, total phenolic content and DPPH (1,1-diphenyl-2-picrylhydrazyl radical) concentration. The activities of antioxidant enzymes, namely superoxide dismutase (SOD), catalase (CAT) and peroxidase (POX), were also reduced under salt stress. However, MDA concentration was decreased, while H 2 O 2 and proline content did not drastically change under the stress conditions caused by NaCl. Exogenous application of SNP altered the biochemical parameters of centaury shoots grown under salt stress. In this case, increased photosynthetic pigment content, total phenolics and proline content were noted, with reduced MDA, but not H 2 O 2 , concentration was observed. In addition, the exogenous application of SNP increased the degree of DPPH reduction as well as SOD, CAT and POX activities.

The rapid production and numerous applications of nanomaterials warrant the necessity and importance of examining nanoparticles in terms to their environmental and biological effects and implications. In this study, the effects of a water-soluble hyper-harmonized hydroxyl-modified fullerene (3HFWC) on cherry tomato seed germination, seedlings growth, physiological response and fruiting was evaluated. Changes in the photosynthetic pigments content, oxidative stress assessment, and aquaporin genes expression in cherry tomato plants were studied after during short- and long-term continuous exposure to 3HFWC nanosubstance (200 mg/L). Increased levels of photosynthetic pigments in leaves, lycopene in fruits, decreased levels of hydrogen peroxide content, activation of cellular antioxidant enzymes such as superoxide dismutase, catalase and peroxidase and increased aquaporin gene expression (PIP1;3 , PIP1;5 and PIP2;4 ) were observed in 3HFWC nanosubstance-exposed plants in comparison to control, untreated cherry tomato plants. The 3HFWC nanosubstance showed positive effects on cherry tomato seed germination, plantlet growth and lycopene content in fruits and may be considered as a promising nanofertilizer.

Plant hormones regulate numerous developmental and physiological processes. Abiotic stresses considerably affect production and distribution of phytohormones as the stress signal triggers. The homeostasis of plant hormones is controlled by their de novo synthesis and catabolism. The aim of this work was to analyse the contents of total and individual groups of endogenous cytokinins (CKs) as well as indole-3-acetic acid (IAA) in AtCKX overexpressing centaury plants grown in vitro on graded NaCl concentrations (0, 50, 100, 150, 200 mM). The levels of endogenous stress hormones including abscisic acid (ABA), salicylic acid (SA) and jasmonic acid (JA) were also detected. The elevated contents of total CKs were found in all analysed centaury shoots. Furthermore, increased amounts of all five CK groups, as well as enhanced total CKs were revealed on graded NaCl concentrations in non-transformed and AtCKX roots. All analysed AtCKX centaury lines exhibited decreased amounts of endogenous IAA in shoots and roots. Consequently, the IAA/bioactive CK forms ratios showed a significant variation in the shoots and roots of all AtCKX lines. In shoots and roots of both non-transformed and AtCKX transgenic centaury plants, salinity was associated with an increase of ABA and JA and a decrease of SA content., (© 2021. The Author(s).)

Duckweed ( L. minor ) is a cosmopolitan aquatic plant of simplified morphology and rapid vegetative reproduction. In this study, an H. paralvei bacterial strain and its influence on the antioxidative response of the duckweeds to phenol, a recalcitrant environmental pollutant, were investigated. Sterile duckweed cultures were inoculated with H. paralvei in vitro and cultivated in the presence or absence of phenol (500 mg L -1 ), in order to investigate bacterial effects on plant oxidative stress during 5 days. Total soluble proteins, guaiacol peroxidase expression, concentration of hydrogen peroxide and malondialdehyde as well as the total ascorbic acid of the plants were monitored. Moreover, bacterial production of indole-3-acetic acid (IAA) was measured in order to investigate H. paralvei's influence on plant growth. In general, the addition of phenol elevated all biochemical parameters in L. minor except AsA and total soluble proteins. Phenol as well as bacteria influenced the expression of guaiacol peroxidase. Different isoforms were associated with phenol compared to isoforms expressed in phenol-free medium. Considering that duckweeds showed increased antioxidative parameters in the presence of phenol, it can be assumed that the measured parameters might be involved in the plant's defense system. H. paralvei is an IAA producer and its presence in the rhizosphere of duckweeds decreased the oxidative stress of the plants, which can be taken as evidence that this bacterial strain acts protectively on the plants during phenol exposure.

In ornamental geophytes, conventional vegetative propagation is not economically feasible due to very slow development and ineffective methods. It can take several years until a new plant is formed and commercial profitability is achieved. Therefore, micropropagation techniques have been developed to increase the multiplication rate and thus shorten the multiplication and regeneration period. The majority of these techniques rely on the formation of new bulbs and their sprouting. Dormancy is one of the main limiting factors to speed up multiplication in vitro. Bulbous species have a period of bulb dormancy which enables them to survive unfavorable natural conditions. Bulbs grown in vitro also exhibit dormancy, which has to be overcome in order to allow sprouting of bulbs in the next vegetation period. During the period of dormancy, numerous physiological processes occur, many of which have not been elucidated yet. Understanding the process of dormancy will allow us to speed up and improve breeding of geophytes and thereby achieve economic profitability, which is very important for horticulture. This review focuses on recent findings in the area of bulb dormancy initiation and release in fritillaries, with particular emphasis on the effect of plant growth regulators and low-temperature pretreatment on dormancy release in relation to induction of antioxidative enzymes' activity in vitro.

Centaurium erythraea (centaury) is a traditionally used medicinal plant, with a spectrum of secondary metabolites with confirmed healing properties. Centaury is an emerging model in plant developmental biology due to its vigorous regenerative potential and great developmental plasticity when cultured in vitro. Hereby, we review nearly two decades of research on somatic embryogenesis (SE) in centaury. During SE, somatic cells are induced by suitable culture conditions to express their totipotency, acquire embryogenic characteristics, and eventually give rise to somatic embryos. When SE is initiated from centaury root explants, the process occurs spontaneously (on hormone-free medium), directly (without the callusing phase), and the somatic embryos are of unicellular origin. SE from leaf explants has to be induced by plant growth regulators and is indirect (preceded by callusing). Histological observations and culture conditions are compared in these two systems. The changes in antioxidative enzymes were followed during SE from the leaf explants. Special focus is given to the role of arabinogalactan proteins during SE, which were analyzed using a variety of approaches. The newest and preliminary results, including centaury transcriptome, novel potential SE markers, and novel types of arabinogalactan proteins, are discussed as perspectives of centaury research.

The bulb is the main propagation organ of snake's head fritillary ( Fritillaria meleagris L.), a horticulturally attractive and rare geophyte plant species. In this study, we investigated the effect of soaking bulbs in GA 3 solution (1, 2, and 3 mg L -1 ) combined with low-temperature treatment (7 °C) on breaking the dormancy of in vitro bulbs. Sugar status (total soluble sugars, glucose, and fructose content) was analyzed in different parts of the sprouted bulbs. The results showed that the soluble sugar concentration was highest in bulbs soaked in GA 3 . The main sugar in fritillary bulbs was glucose, while fructose content was much lower. Glucose concentration dramatically increased after bulb chilling (7 °C), and its accumulation was predominantly detected in the lower sprout portion during the first weeks of sprouting. Sugar concentration was significantly lower in nonchilled bulbs, which indicates the importance of low temperature in bulb development and sprouting.

This study was carried out to examine the drought effect on development, physiological, biochemical and molecular parameters in Impatiens walleriana grown ex vitro. Experiment design included three treatments: Control plants-grown under optimal watering (35%-37% of soil moisture content), drought-stressed plants-non-irrigated to reach 15% and 5% of soil moisture content and recovery plants-rehydrated for four days to reach optimal soil moisture content. Drought reduced fresh weight, total leaf area, as well as dry weight of I. walleriana shoots. Drought up-regulated expression of abscisic acid (ABA) biosynthesis genes 9-cis-epoxycarotenoid dioxygenase 4 ( NCED4 ) and abscisic aldehyde oxidase 2 ( AAO2 ) and catabolic gene ABA 8'-hydroxylase 3 ( ABA8ox3 ) which was followed by increased ABA content in the leaves. Decrement in water potential of shoots during the drought was not accompanied with increased amino acid proline content. We detected an increase in chlorophyll, carotenoid, total polyphenols and flavonols content under drought conditions, as well as malondialdehyde, hydrogen peroxide and DPPH (1,1'-diphenyl-2-picrylhydrazyl) activity. Increased antioxidant enzyme activities (superoxide dismutase, peroxidase and catalase) throughout drought were also determined. Recovery treatment was significant for neutralizing drought effect on growth parameters, shoot water potential, proline content and genes expression.

Bulbs are the main vegetative reproductive organs of Fritillaria meleagris L. In nature, as well as in vitro, they become dormant and require low temperatures for further growth during the next vegetative period. In the present study, using 10 μM of gibberellic acid (GA 3 ), or gibberellin biosynthesis (GA) inhibitors-ancymidol (A) and paclobutrazol (P)-the dynamic changes in soluble sugars, fructose and glucose content, fresh weight and sprouting capacity were investigated. F. meleagris bulbs were cultured on medium with GA 3 and GA inhibitors for 1, 2 and 5 weeks at two different temperatures (24 and 7 °C). GA 3 improved bulb fresh weight, as well as sprouting percentage at both tested temperatures, compared to the control. The highest fresh weight increase (57.7%) and sprouting rate (29.02%) were achieved when bulbs were grown at 24 °C for 5 weeks. In addition, soluble sugar content was the highest in bulbs grown for 5 weeks on medium supplemented with GA 3 . The main sugar in fritillary bulbs was glucose, while fructose content was lower. The sensitivity of bulbs to GA inhibitors differed and significantly affected sugar content in bulbs. To our knowledge, this is the first study of the sugar composition in F. meleagris bulbs during breaking of the bulb's dormancy and its sprouting.

The main topic of this study is the bioremediation potential of the common duckweed, Lemna minor L., and selected rhizospheric bacterial strains in removing phenol from aqueous environments at extremely high initial phenol concentrations. To that end, fluorescence microscopy, MIC tests, biofilm formation, the phenol removal test (4-AAP method), the Salkowski essay, and studies of multiplication rates of sterile and inoculated duckweed in MS medium with phenol (200, 500, 750, and 1000 mg L -1 ) were conducted. Out of seven bacterial strains, six were identified as epiphytes or endophytes that efficiently removed phenol. The phenol removal experiment showed that the bacteria/duckweed system was more efficient during the first 24 h compared to the sterile duckweed control group. At the end of this experiment, almost 90% of the initial phenol concentration was removed by both groups, respectively. The bacteria stimulated the duckweed multiplication even at a high bacterial population density (>10 5 CFU mL -1 ) over a prolonged period of time (14 days). All bacterial strains were sensitive to all the applied antibiotics and formed biofilms in vitro. The dual bacteria/duckweed system, especially the one containing strain 43- Hafnia paralvei C32-106/3, Accession No. MF526939, had a number of characteristics that are advantageous in bioremediation, such as high phenol removal efficiency, biofilm formation, safety (antibiotic sensitivity), and stimulation of duckweed multiplication.

Morphogenesis in vitro is a complex and still poorly defined process. We investigated esterase and peroxidase isoforms detected in bulb scale, during Fritillaria meleagris morphogenesis. Bulbs were grown either at 4 °C or on a medium with an increased concentration of sucrose (4.5%) for 30 days. After these pre-treatments, the bulb scales were further grown on nutrient media that contained different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or thidiazuron (TDZ). Regeneration of somatic embryos and bulblets occurred at the same explant. The highest numbers of somatic embryos and bulblets were regenerated on the medium containing 2,4-D and KIN (1mg/L each), while morphogenesis was most successful at a TDZ concentration between 0.5 and 1mg/L. Monitoring of esterases and peroxidases was performed by growing bulb scales on a medium enriched with 2,4-D and KIN or TDZ (1mg/L), and the number and activity of isoforms were followed every 7 days for 4 weeks. In control explants, six isoforms of esterase were observed. Three isoforms of peroxidase were not detected in the control bulb scale, which has not begun its morphogenesis process., (Copyright © 2015 Académie des sciences. Published by Elsevier SAS. All rights reserved.)