Interaction ammonium-nitrate: Response to oxidative stress in chicory plants

The aim of this work was to study, as a function of the different availability of nitrogen in the reduced form, mineral and organic, the induction of the synthesis of some ROS-scavenging molecules and the evolution of some enzymatic activities such as ascorbate peroxidase (APX) and polyphenoloxidase (PPO). 
Chicory seedlings were grown in nutritive solution for 35 days in controlled conditions. On the 14th day, one third of the plants was transferred into a nutritive solution containing (NH4)2SO4 60 mM, one third was transferred into a medium containing Urea 60 mM, and the remaining was let grow into the nutrition solution, as a control. Three samplings of leaves were performed, respectively after 21, 28 and 35 days of growth.
The urea and ammonium sulphate-treated samples showed higher ascorbic acid and polyphenol contents than the control, together with a lower anthocyanins content. APX showed the highest activity in the urea-treated samples, while the highest PPO activity was to refer to samples treated with ammonium sulphate.
The variations of the organic components showed the incidence of the nitrogen supply in the reduced form on the cell redox potential, confirming the importance of fertilization for obtaining high amounts of antioxidant molecules.
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Differential dynamics of flavonoid biosynthesis and accumulation in six medicinal herbs under full sunlight exposure

The dynamics of epidermal flavonoid (epFlav) induction was monitored during 11 days in the leaves of three Ocimum basilicum varieties: Americanum, Genovese and Purpurescens, Salvia officinalis, Eruca sativa and Verbascum thapsus previously grown in a glasshouse (receiving 16% of sunlight irradiance without UV). At full sunlight plants received mean daily doses of 6.97 MJ m-2, 0.86 MJ m-2 and 22.46 kJ m-2 in the photosynthetically active radiation, UV-A and UV-B wavelengths, respectively. Although constitutive concentration of epFlav was similar, dynamics of their induction was species-specific. Significant increase in epFlav accumulation was observed 30h after exposure to sunlight in all species except O. basilicum var. Purpurescens, in which the increase was delayed. This delay was correlated with induction of constitutive epidermal anthocyanins. Total leaf flavonoids were unchanged, in contrast to epidermal, emphasizing their UV-B absorbing function. Total antioxidative capacity increased at least 2-fold under sunlight, which was in accordance with total phenolic increase. Leaf dry biomass was 50-100% higher in sunlight-exposed compared to glasshouse grown plants, while fresh biomass was unchanged, indicative of leaf thickening. We showed that flavonoids accumulation was dependent on light conditions and constitutive pool of UV-B absorbing compounds

Influence of UV radiation on the content of secondary metabolites in tomato grown in different environmental conditions

In this study we investigated the effects of ambient ultraviolet (UV, 280- 400 nm) radiation on the composition of secondary metabolites of high nutritional value (pigments and flavonoids) in Lycopersicon esculentum (tomato) fruits. Tomato plants were grown during summer in the open field and two types of polytunnels, PT1 ( UV-A 1.45 mV cm-2, UV-B 3.84 μV cm-2, PAR 750 μmol m-2 s-1) and PT2 (UV-A 0.37 mV cm-2, UV-B 0.03 μV cm-2, PAR 760 μmol m-2 s-1) and fruits were taken in august. The contents of lycopene, β-carotene and flavonoids (quercetin and kaempferol) were determined by HPLC in the tomato exocarp and pulp. Regardless of UV radiation exposure, higher amounts of lycopene, flavonoids and β-carotene were measured in exocarp compared to the pulp. Accumulation of phenolics, in both exocarp and pulp was the highest in fruits collected in the field. Similarly, the concentration of epidermal flavonoids was the highest in the leaves of plants from the open field. These results support the protective functions of flavonoids as UV-screeners and antioxidants from high PAR. Moreover, the content of lycopene was the highest in exocarp and β-carotene in the pulp of fruits exposed to full ambiental UV radiation doses, compared to fruits collected in the polytunells. The results implicate that the controlled exposure to solar UV radiation during crop growth may be used as a stimulator of biosynthesis of compounds with high antioxidative capacity thus improving the fruit quality and nutritional value

Twenty different late embryogenesis abundant proteins (LEAPs) accumulate in desiccated Ramonda serbica leaves

Resurrection plant Ramonda serbica Panc. survives desiccation for a long period and fully recovers metabolic functions already within one day upon watering [1]. Besides osmotic stress, desiccation provokes the accelerated generation of reactive oxygen species. The aim of our study was obtaining more insight into the mechanisms of desiccation tolerance in R. serbica by TMT labelled comparative quantitative proteomics of hydrated (HL) and desiccated leaves (DL). After de novo transcriptome analysis, 189456 transcripts with 189003 unigenes were annotated with seven common databases. Proteomic analysis allowed for the relative quantification of 895 different protein groups, 321 with a statistically significant difference in abundance between FL and DL. Among them, 25% referred to chloroplast and almost the same percentage were associated with desiccation and oxidative stress. Almost all differentially abundant proteins related to photosynthetic processes were down-regulated in DL, while those required for protein translation were more abundant in HL. Within differentially abundant proteins involved in antioxidative defence, the levels of enzymes involved in ascorbate-glutathione cycle, peroxiredoxins, Fe and Mn superoxide dismutase (SOD) were all reduced in DL, while germin-like proteins, three Cu/Zn SOD isoforms and polyphenol oxidases were more abundant in DL compared with HL. The protein family with the highest number of members showing the greatest accumulation upon desiccation comprised twenty different late embryogenesis abundant proteins (LEAPs), similarly as found by differential transcriptomic analysis. Taken together, our results imply a key role of LEAPs and Cu/Zn SOD in protective mechanism against desiccation in R. serbica, that may have significant implications on drought-related studies of crops grown in arid areas. This work was supported by the Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI, grant number 6039663). M.V. wishes to acknowledge the support of COST Action BM1405 for approving STSM in Padua during 2017 and 2018.Related to poster presentation: [https://cherry.chem.bg.ac.rs/handle/123456789/4424

Karakterizacija i odabir autohtonih bakterija mliječno-kiselog vrenja kao starter kultura u zanatskoj proizvodnji vlasinskog sira iz svježeg kozjeg mlijeka

The aim of this study is the isolation, characterization and identification of autochthonous lactic acid bacteria (LAB) from artisanal Vlasina raw goat\u27s milk cheese for the selection of potential starter cultures. Soft white Vlasina cheese was manufactured at a household on the Stara Planina Mountain using traditional techniques without starter cultures. One hundred and forty nine LAB isolates were collected from two samples of Vlasina cheese, designated as BGVL2 (5 days old) and BGVL2a (15 days old). The population of LAB in the cheese samples was characterized by phenotype-based assays and presumptively identified using repetitive element palindromic polymerase chain reaction (rep-PCR) with the primer (GTG)5. Results were confirmed by 16S rDNA sequencing. Among the BGVL2 isolates (56), the most numerous LAB species were Leuconostoc pseudomesenteroides (27) and Lactococcus lactis (26). In 15-day-old BGVL2a (93 isolates), Lactobacillus plantarum (33), Enterococcus durans (26) and Pediococcus pentosaceus (14) were predominant. Lc. lactis ssp. lactis BGVL2-8 showed good acidification ability and the ability to produce antimicrobial compounds, Lb. plantarum BGVL2a-18 had good proteolytic ability and produced exopolysaccharides, while BGVL2-29 and BGVL2-63, which belonged to the species Ln. pseudomesenteroides, utilized citrate and produced diacetyl and acetoin. They appeared to be suitable candidates for inclusion in the starter culture. This study contributed to the understanding of the role of autochthonous LAB in the quality of artisanal cheese and the possibility of using the selected LAB as potential starter cultures for cheese making under controlled conditions.Svrha je ovoga rada bila izolirati, karakterizirati i identificirati autohtone bakterije mliječno-kiselog vrenja koje se mogu upotrijebiti kao starter kulture u zanatskoj proizvodnji vlasinskog sira iz svježeg kozjeg mlijeka. Meki je bijeli vlasinski sir bio izrađen u domaćinstvu na Staroj Planini tradicionalnim postupkom bez dodatka starter kultura. Iz dvaju je uzoraka vlasinskih sireva, označenih kao BGVL2 (sir star 5 dana) i BGVL2 (sir star 15 dana) prikupljeno 149 izolata bakterija mliječno-kiselog vrenja. Populacija je bakterija iz uzoraka sireva okarakerizirana pomoću fenotipskih testova i primarno identificirana primjenom metode rep-PCR s početnicom (GTG)5. Rezultati su potvrđeni sekvenciranjem 16S rDNA. Među izolatima iz sira BGVL2 (njih 56) najbrojnije su bile bakterije Leuconostoc pseudomesenteroides (27) i Lactococcus lactis (26). U siru BGVL2a (93 izolata), starom 15 dana, prevladavale su vrste Lactobacillus plantarum (33), Enterococcus durans (26) i Pediococcus pentosaceus (14). Izolat Lc. lactis ssp. lactis BGVL2-8 pokazao je dobru sposobnost zakiseljavanja i proizvodnje antimikrobnih spojeva. Izolat Lb. plantarum BGVL2a-18 imao je dobru proteolitičku aktivnost i sposobnost proizvodnje egzopolisaharida, dok su izolati BGVL2-29 i BGVL2-63 vrste Ln. pseudomesenteroides za rast koristili citrate, a proizvodili diacetil i acetoin. Stoga su navedeni izolati odabrani kao starter kulture. Ovo je istraživanje pridonijelo razumijevanju uloge autohtonih bakterija mliječno-kiselog vrenja u zanatskoj proizvodnji kvalitetnog sira, te je potvrdilo mogućnost primjene odabranih izolata kao starter kultura pri izradi vlasinskog sira u kontroliranim uvjetima

Twenty different late embryogenesis abundant proteins (LEAPs) accumulate in desiccated Ramonda serbica leaves

Resurrection plant Ramonda serbica Panc. survives desiccation for a long period and fully recovers metabolic functions already within one day upon watering [1]. Besides osmotic stress, desiccation provokes the accelerated generation of reactive oxygen species. The aim of our study was obtaining more insight into the mechanisms of desiccation tolerance in R. serbica by TMT labelled comparative quantitative proteomics of hydrated (HL) and desiccated leaves (DL). After de novo transcriptome analysis, 189456 transcripts with 189003 unigenes were annotated with seven common databases. Proteomic analysis allowed for the relative quantification of 895 different protein groups, 321 with a statistically significant difference in abundance between FL and DL. Among them, 25% referred to chloroplast and almost the same percentage were associated with desiccation and oxidative stress. Almost all differentially abundant proteins related to photosynthetic processes were down-regulated in DL, while those required for protein translation were more abundant in HL. Within differentially abundant proteins involved in antioxidative defence, the levels of enzymes involved in ascorbate-glutathione cycle, peroxiredoxins, Fe and Mn superoxide dismutase (SOD) were all reduced in DL, while germin-like proteins, three Cu/Zn SOD isoforms and polyphenol oxidases were more abundant in DL compared with HL. The protein family with the highest number of members showing the greatest accumulation upon desiccation comprised twenty different late embryogenesis abundant proteins (LEAPs), similarly as found by differential transcriptomic analysis. Taken together, our results imply a key role of LEAPs and Cu/Zn SOD in protective mechanism against desiccation in R. serbica, that may have significant implications on drought-related studies of crops grown in arid areas. This work was supported by the Science Fund of the Republic of Serbia (PROMIS project LEAPSyn-SCI, grant number 6039663). M.V. wishes to acknowledge the support of COST Action BM1405 for approving STSM in Padua during 2017 and 2018

ACCLIMATION OF PEDUNCULATE OAK SEEDLINGS TO DIFFERENT LIGHT CONDITIONS IN THE FIRST MONTHS AFTER GERMINATION

The first months are the most sensitive phase of oak seedling development. Light conditions have an important role in this sense, both from the physiological and management viewpoint. We investigated the response of pedunculate oak seedlings to three growth light intensities (100, 550 and 2000 μmol cm−2s−1) during development of the first and second growth flush. The low and high light intensities of PAR are supposed to mimic the effects of solar radiation under extreme natural conditions (closed canopy and open field). The response of seedlings to different light intensities was evaluated by determining the photochemical activity of photosystem II, leaf chlorophyll concentration and epidermal flavonoid accumulation for both growth flushes. At the end of the experiment (after 4.5 months) the effects of different treatments on growth parameters were also determined. We showed here that oak seedlings responded to varying light intensities by modifying their physiological and morphological traits of successive growth flushes. At medium light, seedlings had the highest PSII photochemical activity in the 2nd flush. High light induced very low photochemical efficiency of photosystem II in both growth flushes indicating the development of high non-photochemical quenching of chlorophyll fluorescence as part of photoprotective mechanism. In accordance with high photosynthetic yield at ML, the investment of photosynthates in growth, especially biomass allocation towards root system was confirmed. ML was optimal for seedling development in the first months. The results may contribute to a better understanding of oak seedling development and acclimation and could have importance for oak natural regeneration

Identification of phenolic compounds in cecidogen and Linaria vulgaris. Changes in phenolic metabolism during gall formation induced by Rhinusa pilosa

Rhinusa pilosa (Gyllenhaal) (Coleoptera, Curculionidae) induces galls in Linaria vulgaris Mill., Yellow or common toadflax (Plantaginaceae). Gall is defined as atypical plant growth induced by host-specific organisms, and represents larval chamber which provides food and protection during insect development. It has been shown that gall formation is triggered by ovoipositional fluid (cecidogen). In order to determine which cecidogen compounds may have bioactive properties, we performed comparative analysis of phenolics profiles of cecidogen and stem extracts of L. vulgaris using UHPLC coupled with OrbiTrap mass analyzer. In addition, we analyzed the effects of feeding, oviposition and early gall development on Class III peroxidase activity (POD, EC 1.11.1.7) and phenolic content in the stems of L. vulgaris during seven days. Out of 55 phenolic compounds identified in cecidogen and L. vulgaris stem, one unknown phenolic glycoside was found only in cecidogen, which produced an MS2 base peak at 387 m/z, and 327 m/z and 267 m/z base peaks at MS3 and MS4 fragmentation, respectively. Gall development during seven days was accompanied by decreased concentrations of hydroxybenzoic, hydroxycinnamic acids and flavonoids and by slight lignin deposition. An oscillatory induction of POD activity, with the first peak obtained 3 h after oviposition, was related to oxidative burst during stem wounding and oviposition. Overall results suggested the importance of phenolics in stem structural changes and regulation of plant metabolism induced by R. pilosa

High PAR and UV-B radiation-induced differential responses in green and white leaf sectors of Pelargonium zonale in relation to sugar, antioxidative and phenolic metabolism

In this study we investigated the specific effects of high photosynthetically active radiation (PAR) and ecologically relevant UV-B radiation (0.90 W m-2) on antioxidative, phenolic and sugar metabolism in variegated Pelargonium zonale plants. The green-white leaf variegation in these plants presents a suitable model system for examining “source-sink” interactions within the same leaf. High PAR (1350 μmol m-2 s-1) and UV-B radiation induced tissue specific responses in variegated P. zonale leaves. While UV-B radiation had a pronounced effect on phenolic content in the white tissue, high PAR intensity stimulated accumulation of phenylpropanoids and flavonoids with preferential antioxidative vs. UV-screening function in green tissue. High PAR stimulated the increase of antioxidative metabolism in both leaf sections. However, the greater enhancement of ascorbate peroxidase and catalase activities and ascorbate content under HL+UV-B than HL only in green sectors, indicated that UV-B radiation and high PAR synergistically stimulated antioxidative defense. These results indicate that green tissue can be considered as high light acclimated, provided with an efficient defense against potential oxidative pressure under high PAR, along with significant protective role of UV-B radiation. Efficient sugar transport from green to white tissue was stimulated by both UV-B radiation and high PAR intensity. By stimulation of starch and sucrose breakdown and carbon allocation in the form of soluble sugars from “source” (green) tissue to “sink” (white) tissue, UV-B radiation stimulates a compensatory mechanism for phenylpropanoid and flavonoid biosynthesis in white tissue, due to the lack of photosynthesis

The usage of silicon fertilisation in order to mitigate the oxidative stress and to improve the resilience of barley subjected to drought

Drought causes huge agricultural and economic losses worldwide. Silicon (Si) is considered abeneficial element for plants. It mitigates stress caused by salinity, drought, and high and low temperaturesby promoting antioxidant production. However, the underlying mechanisms are still notelucidated. We investigated Si fertilisation effect on photosynthetic parameters, trichome number,leaf optical properties, as well as profiles of amino acids and polyphenols in barley exposed towater shortage. Silicon was applied in three growth stages: (i) before the flag leaf emergence;(ii) prior to the grain filling phase; (iii) at the grain filling phase start. Drought negatively impactsphotochemical efficiency, stomatal conductance, photosynthetic pigment content, and leaf reflectiveand transmissive properties. Si application between flag leaf emergence and grain fillinghad the strongest effect on light reflectance. Among all analyzed phenolics, saponarin was themost abundant in all samples, irrespective of water regime and Si supply. Caffeoyl ester was theonly hydroxycinnamic acid showing significant accumulation with the latest applied Si comparedto no added Si upon drought. The major amino acids in barley leaves were glutamate, glutamine,aspartate, asparagine, and serine. Aspartate content was the highest in leaves exposed to droughtwithout Si addition, while lysine was the most accumulated in the leaves supplemented by Si atthe grain filling phase start. Proline was 2.5 times more abundant in the leaves exposed to droughtregardless of Si treatment. Taken together, although Si did not mitigate drought stress effects, itseffect was dependent on the barley growth phase prior to supplementation.Book of Abstracts / 4th International Conference on Plant Biology [and] 23rd SPPS Meeting, 6-8 October 2022, Belgrad