Electrophoretic protein patterns and peroxidase activity related to morphogenesis in Mammillaria gracillis tissue culture

The cactus Mammillaria gracillis Pfeiff. was propagated in vitro on Murashige and Skoog (MS) medium without any growth regulators. At the bases of some plants abundant callus masses developed. Lower agar and higher MS salt concentrations stimulated callus production. In the culture, morphologically normal and hyperhydric shoots were regenerated. The growth of crown-gall tumour was induced on disc-like explants of in vitro grown plants infected with Agrobacterium tumefaciens, B6S3 strain. Calli appeared on both infected and control explants. The tumorous tissue grew more intensively than the control. The transformed character of the callus was confirmed by PCR amplification of a gene 6a of T-DNA. Gene expression in cactus shoots (grown in vitro or in pot), callus, hiperhydric and morphologically normal regenerated shoots and crown-gall tumour were analysed at the level of the electrophoretic pattern of soluble proteins. Some tumour-specific polypeptides were detected (76, 32-39, and 23 kDa). That of 42 kDa was highly expressed in callus and hyperhydric shoots. A faint 58 kDa band was found in all extracts except in the pot-grown shoot. Relatively high peroxidase activity was detected in callus and shoot regenerants and it was lower in tumour and the lowest in plant shoots

CHANGES IN EXPRESSION OF PLANT PROTEINS INDUCED BY SILVER NANOPARTICLES

Nanotehnologija je zadnja u nizu tehnologija koje najavljuju novu eru u napretku društva. Male dimenzije nanočestica (engl. nanoparticles, NPs), s barem jednom dimenzijom između 1 i 100 nm, rezultiraju jedinstvenim kemijskim i fizikalnim značajkama, zbog čega se nanočestice proizvode u velikim količinama za upotrebu u proizvodima za široku potrošnju. Među različitim vrstama dostupnih nanomaterijala dominiraju nanočestice srebra (AgNP), zbog dobro poznatog protubakterijskog i protugljivčnog učinka srebra. Zbog široke upotrebe AgNP, raste zabrinutost glede sigurnosti njihove upotrebe i mogućih štetnih učinaka na okoliš. Iako je do sada objavljeno nekoliko toksikoloških studija na nanočesticama srebra, još uvijek je teško donijeti konačne zaključke o njihovoj toksičnosti. Naime, čim se oslobode u okoliš, započinje njihova transformacija, što mijenja njihova svojstva i time direktno utječe na transport, sudbinu i moguću toksičnost nanosrebra. Kao primarni proizvođači, biljke predstavljaju vitalan dio zdravog ekološkog sustava, ali također imaju i važnu ulogu u transportu i bioakumulaciji toksičnih tvari u hranidbene lance. Toksikološke studije o učincima AgNPs provedene na biljkama su malobrojne i nedorečene. Nadalje, nedostaju i podaci o učincima AgNPs na važne stanične procese. Stoga, u našim istraživanjima želimo otkriti i objasniti toksične učinke nanosrebra na biljke, što će doprinijeti procjeni okolišnog rizika izlaganja nanočesticama srebra, ali i novim saznanjima o mehanizmima toksičnosti. Istraživanja provodimo na luku i duhanu, ekonomski važnim biljnim vrstama koje se često koriste i kao modelni organizmi u istraživanjima abiotičkog stresa. Cilj nam je utvrditi dolazi li tijekom izlaganja različitih razvojnih stadija ovih biljaka nanosrebru do kvalitativnih i kvantitativnih promjena u ekspresiji proteina i definirati osjetljive i selektivne biomarkere toksičnosti nanočestica srebra koji bi se mogli koristiti u biomonitoringu okoliša. Dobiveni rezultati moći će se primijeniti i za procjenu sigurnosti korištenja nanočestica srebra u poljoprivrednim proizvodima, kao što su umjetna gnojiva i pesticidi, koji se sve više koriste u uzgoju ostalih poljoprivrednih vrsta.Nanotechnology is the latest in a long series of technologies which announces new era of technologies-driven prosperity. The small size of nanoparticles (NPs), with at least one dimension between 1 and 100 nm, results in unique chemical and physical characteristics, which is why they are being produced in large quantities for implementation in various consumer products. Among different available NPs, silver nanoparticles (AgNPs) are the dominating nanomaterial because of well-known silver antibacterial and antifungal properties. Due to widespread use and exposure to these nanomaterials, there is ever-growing concern regarding their potential detrimental impacts on the environment. Although there have been quite a number of toxicological studies published on AgNPs, it is still difficult to draw definite conclusions about their toxicity. As soon as AgNPs are released into the environment, they start to transform, which modifies their properties influencing their transport, fate and possible toxicity. As primary producers plants are the vital part of healthy ecosystems but also play a significant role in transport and bioaccumulation of possibly toxic substances into food chains. Toxicological studies of AgNPs conducted on plants are scare and inconclusive. Moreover, there is a lack of information about AgNPs-induced effects on important cellular processes. Therefore, in this research we aim to reveal and explain the toxicity of a set of diverse AgNPs to plants, onion and tobacco, in order to contribute to the environmental hazard assessment of AgNPs as well as to the basic knowledge about the mechanisms of their toxicity. We want to establish the possible proteomic changes upon exposure to treatments with AgNPs. The results will be used to define biomarkers of AgNP-induced phytotoxicity, which could be useful for environmental biomonitoring. Obtained results can also be applied to estimate the possibility of use and safety of AgNPs implemented in agricultural products, such as fertilizers and pesticides

Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

Background and Purpose: Recombinant DNA technology has allowed expression of different heterologous proteins in many host systems, ranging from prokaryotic to eukaryotic organisms. Therapeutic properties of recombinant proteins are very often affected by the composition and heterogeneity of their glycans. Conventional expression systems for recombinant pharmaceuticals typically do not address this problem and result with products that contain a mixture of glycoforms that are neither identical to human glycans nor optimized for enhanced biological activity. Over the last decade plants have been developed as production platforms for recombinant proteins with pharmaceutical or industrial applications. Namely, plant expression systems contain very small differences in the post-translational modifications, mainly glycosylation, which can partly be overpowered by glycoengineering, whose goal is production of recombinant proteins with highly homogenous glycosylation that closely resembles the native system. This review attempts to present current accomplishments in the production of plant-derived glycoconjugates with humanized N- and O-glycans. Materials and Methods: Main goal of N-glycoengineering is to reduce or eliminate plant-specific N-glycans, and at the same time to introduce mammalian-specific N-glycans through the several approaches. The easiest way is to change intracellular targeting of plant-made recombinant proteins and to ensure their retention in the ER; next approach is to eliminate the addition of plant-specific glycans; while the final step is engineering the plant glycosylation pathway to introduce mammalian glycotransferases into plants with generation of biantennary and multi-antennary structures on complex N-glycans. Due to significant differences in O-glycosylation between humans and plants, different approaches to engineering of O-glycosylation have been taken. Besides having their typical O-glycoslyation on Hyp-residues, plants in general miss the machinery for production of mammalian-type O-glycosylation. Attempts have been made to mimic mammalian O-glycosylation in plants, specifically the mucin-type addition of GalNAc residues. Result: Efficient generation of bisected tetraantennary complex N-glycans without typical plant glycoepitopes on human erythropoietin (hEPO) and human transferrin (hTF) was obtained in Nicotiana benthamiana plants, thus demonstrating generation of recombinant proteins with human-type N-glycosylation at great uniformity. As for the O-glycosylation, attempts to produce mucin-type O-GalNAc and disialylated core 1 O-linked glycan structures on hEPO in N. benthamiana transgenic plants proved to be successful. Moreover, although small amounts of Hyp residues were found on recombinant EPO, no plant-specific O-glycans were detected, which demonstrates that plants are eligible candidates for production of recombinant therapeutics with fully humanized O- and N-glycans. Conclusion: Plants and methods of plant molecular farming offer a powerful expression platform for the production of a variety of recombinant proteins, which show similar, or even higher, biological activity then protein or native homologs in cultured mammalian cells currently used for large-scale production

Developmentally specific soluble and membrane proteins and glycoproteins in Mammillaria gracillis Pfeiff. (Cactaceae) tissue culture

Artificial environmental conditions in the tissue culture influence growth and induce aberration from the characteristic organization pattern. As CAM plants, cacti are particularly susceptible to altered growth environment. In vitro propagated Mammillaria plants spontaneously produce callus which regenerates normal and hyperhydric shoots without the addition of any growth regulator. In order to compare habituated callus with tumour, cactus cells were transformed with A. tumefaciens. Cactus tumour has never expressed any organogenic potential. The aim of this work was to detect changes in protein and glycoprotein profiles in different developmental stages of Mammillaria tissues. Proteins were separated by SDS-PAGE and silver stained. Glycoproteins were detected according to their affinity to Con A, while the glycan components were further characterized with lectins GNA, DSA, MAA, PNA and SNA. Only few morphogenesis-specific polypeptides were observed on the gels. Differences were more pronounced in the glycoprotein pattern. The greatest number of glycosylated proteins in all cactus tissues was detected by Con A, according to which all untransformed tissues were characterized by the presence of 40 kDa glycoprotein, while the tumor exhibited some specific protein bands. No signal was observed with DSA, while GNA-, PNA-, MAA- and SNA-profiles partially correlated with those detected with Con A. The results demonstrated changes in protein glycosylation related to disarrangement or loss of characteristic tissue organization pattern in Mammillaria gracillis tissue culture

Proteinski i glikoproteinski biljezi morfogeneze u kulturi tkiva kaktusa Mammillaria gracillis Pfeiff.

As plants with Crassulacean Acid Metabolism (CAM), cacti are highly affected by artificial environmental conditions in tissue culture. Plants of Mammillaria gracillis Pfeiff. (Cactaceae) propagated in vitro produced callus spontaneously. This habituated callus regenerated normal and hyperhydric shoots without the addition of growth regulators. In order to compare habituated callus with the tumorous one, cactus cells were transformed with two strains of Agrobacterium tumefaciens: the wild strain B6S3 (tumour line TW) and the rooty mutant GV3101 (tumour line TR). Gene expression in cactus plants, habituated callus, regenerated shoots and two tumour lines was analysed at the level of cellular and extracellular protein and glycoprotein profiles. Proteins were separated by SDS-polyacrylamide gel electrophoresis and 2-D PAGE electrophoresis and silver stained. Concavalin A-peroxidase staining detected glycoproteins with D-manose in their glycan component on protein blots. Developmentally specific protein patterns of Mammillaria gracillis tissue lines were detected. The 2-D PAGE electrophoresis revealed some tissue specific protein groups. The cellular glycoprotein of 42 kDa detected by ConA was highly expressed in undifferentiated tissues (habituated callus, TW and TR tumours) and in hyperhydric regenerants. Tumours produced extracellular proteins of 33, 23 and 22 kDa. The N glycosylation of cellular and extracellular proteins was related to specific developmental stage of cactus tissue

Proteomika u istraživanjima toksičnosti teških metala u biljaka

Plants endure a variety of abiotic and biotic stresses, all of which cause major limitations to production. Among abiotic stressors, heavy metal contamination represents a global environmental problem endangering humans, animals, and plants. Exposure to heavy metals has been documented to induce changes in the expression of plant proteins. Proteins are macromolecules directly responsible for most biological processes in a living cell, while protein function is directly influenced by posttranslational modifications, which cannot be identified through genome studies. Therefore, it is necessary to conduct proteomic studies, which enable the elucidation of the presence and role of proteins under specific environmental conditions. This review attempts to present current knowledge on proteomic techniques developed with an aim to detect the response of plant to heavy metal stress. Significant contributions to a better understanding of the complex mechanisms of plant acclimation to metal stress are also discussed.Biljke su neprestano izložene različitim čimbenicima abiotičkog i biotičkog stresa koji nepovoljno utječu na njihovu produktivnost. Teški metali kao česti zagađivači okoliša vrlo nepovoljno djeluju na sva živa bića, uključujući biljke, životinje i ljude. Poznato je da teški metali mogu mijenjati ekspresiju biljnih proteina. Proteine ubrajamo u biološki vrlo važne makromolekule čija je aktivnost u stanici izravno ovisna o posttranslacijskim modifikacijama, koje nije moguće pratiti na razini genoma. Stoga je nužno provoditi proteomska istraživanja kako bi se razotkrila prisutnost i uloga proteina u različitim vrstama okolišnog stresa. U ovom radu sažete su različite tehnike i metode istraživanja učinaka teških metala na biljni proteom, uključujući i sažet osvrt na složene mehanizme odgovora biljke na stres izazvan teškim metalima

Proteomika u istraživanjima toksičnosti teških metala u biljaka

Plants endure a variety of abiotic and biotic stresses, all of which cause major limitations to production. Among abiotic stressors, heavy metal contamination represents a global environmental problem endangering humans, animals, and plants. Exposure to heavy metals has been documented to induce changes in the expression of plant proteins. Proteins are macromolecules directly responsible for most biological processes in a living cell, while protein function is directly influenced by posttranslational modifications, which cannot be identified through genome studies. Therefore, it is necessary to conduct proteomic studies, which enable the elucidation of the presence and role of proteins under specific environmental conditions. This review attempts to present current knowledge on proteomic techniques developed with an aim to detect the response of plant to heavy metal stress. Significant contributions to a better understanding of the complex mechanisms of plant acclimation to metal stress are also discussed.Biljke su neprestano izložene različitim čimbenicima abiotičkog i biotičkog stresa koji nepovoljno utječu na njihovu produktivnost. Teški metali kao česti zagađivači okoliša vrlo nepovoljno djeluju na sva živa bića, uključujući biljke, životinje i ljude. Poznato je da teški metali mogu mijenjati ekspresiju biljnih proteina. Proteine ubrajamo u biološki vrlo važne makromolekule čija je aktivnost u stanici izravno ovisna o posttranslacijskim modifikacijama, koje nije moguće pratiti na razini genoma. Stoga je nužno provoditi proteomska istraživanja kako bi se razotkrila prisutnost i uloga proteina u različitim vrstama okolišnog stresa. U ovom radu sažete su različite tehnike i metode istraživanja učinaka teških metala na biljni proteom, uključujući i sažet osvrt na složene mehanizme odgovora biljke na stres izazvan teškim metalima

Sugar beet cells’ cellular and extracellular events taking place in response to drought and salinity

Salt and drought stress are important abiotic factors that negatively affect plant growth and yield. To understand how these stress factors affect metabolism at the cellular level, we analyzed cation concentrations and expression of cellular and extracellular proteins, as well as their functions and types. Cells of the industrially important halophyte sugar beet were exposed to 300 mM NaCl and 600 mM mannitol as stressors in modified Gamborg B5 liquid nutrient medium (PG0). Severe stress altered the intracellular concentrations of most of the measured cations. The cellular proteome revealed that both stressors provoked significant differential regulation of 110 cellular proteins. About 80% of the identified proteins were classified in metabolism, energy, or cell rescue, defense and virulence categories. We identified several novel proteins that respond to stress, including a member of the bZIP family of transcription factors, a member of the glycine-rich RNA-binding proteins, and the K+ channel beta subunit. Among extracellular proteins we found previously unreported stress-responsive proteins, a beta-xylosidase and an isoform of chitinase. The obtained results indicate that salt and drought stress disturbed the concentrations of cellular cations and affected the expression of cellular and extracellular proteins in sugar beet cells

Silver nanoparticles affect germination and photosynthesis in tobacco seedlings

Extensive commercialization of silver nanoparticles (AgNPs) raises the risk of their accumulation in the soil-plant system. Once released into the environment, AgNPs are prone to chemical transformations, which make it hard to determine whether their phytotoxic effects are purely NP-related or a consequence of released Ag+ ions. In this study the effects of 25, 50, 75, 100 and 150 μM AgNPs and AgNO3 on seed germination and early growth of tobacco (Nicotiana tabacum L.) seedlings were compared. Additionally, the effects on photosynthetic performance and pigment content were investigated. Germination rate and index values indicated delayed and slower germination in some AgNP treatments. Lower AgNP concentrations stimulated root growth, but induced a prominent reduction in fresh weight. By contrast, all AgNO3 concentrations inhibited root growth but only the higher ones decreased fresh weight. Obtained results imply that the observed AgNP toxicity could be ascribed to NP form and can be correlated with high AgNP stability in the solid medium. On the other hand, the majority of AgNP and AgNO3 treatments induced an increase in chlorophyll content that was accompanied by significantly lower values of relative electron transport rate and coefficient of photochemical quenching, implying an inhibition of the electron transport chain. A similar impact of AgNPs and AgNO3 on photosynthesis can be correlated with lower stability of AgNPs in a liquid medium, resulting in AgNP aggregation and dissolution of Ag+ ions

Oksidacijski stres u tkivu hrena (Armoracia lapathifolia Gilib.) uzgojenom u uvjetima in vitro

In a previous study it was reported that transformed tissue of horseradish (Armoracia lapathifolia Gilib.), obtained by infection of leaf explants with A. tumefaciens, developed two tumour lines with different morphology. One line grew as a completely unorganized tissue (TN – tumour tissue), while the other line grew as a partially organized teratogenous tumour with malformed hyperhydric shoots (TM – teratoma tissue), but did not regenerate the whole plant of normal morphology. The factor responsible for this problem could be the increased production of reactive oxygen species (ROS). Therefore, in this study a possible involvement of activated oxygen metabolism in dedifferentiation and hyperhydricity in TM and TN tissues is investigated. Elevated values of malondialdehyde and protein carbonyl contents found in TM and TN, in comparison with plantlet leaf, confirm the presence of oxidative stress. However, lower H2O2 content was measured in TM and TN. Lipoxygenase (LOX) activity was more pronounced in TM and especially in TN compared to leaf, which suggests that the LOX-dependent peroxidation of fatty acids might be one of the causes of oxidative damage. Moreover, significantly higher peroxidase (PRX) and ascorbate peroxidase (APX) activity as well as the increased number of their isoforms was found in transformed TM and TN in comparison with leaf. On the other hand, significantly lower superoxide dismutase (SOD) activity was found in TM and TN, which correlates with lower H2O2 content. High catalase (CAT) activity measured in leaf and partially organized TM is consistent with the role of CAT in growth and differentiation. In conclusion, in horseradish transformed tissues that underwent dedifferentiation and hyperhydricity, prominent oxidative damage was found. This result suggests that oxidative stress could be associated with the inability of partially organized teratogenous TM to regenerate plantlets with normal morphology.Dosadašnja istraživanja autora pokazala su da je transformirano tkivo hrena (Armoracia lapathifolia Gilib.), nakon infekcije bakterijom A. tumefaciens, razvilo dvije morfološki različite linije tumorskog tkiva. Jedna je linija rasla kao potpuno neorganizirano tumorsko tkivo, a druga kao djelomično organizirani teratogeni tumor s malformiranim hiperhidriranim izdancima (teratomska linija), ali bez mogućnosti regeneriranja biljke normalnih morfoloških karakteristika. Budući da bi povećana proizvodnja reaktivnih oblika kisika mogla uzrokovati promjenu morfoloških karakteristika, u ovom je radu istraživan mogući utjecaj kisika na dediferencijaciju i hiperhidriranost u potpuno neorganiziranom tumorskom tkivu i teratomskoj liniji. Uspoređujući povećane vrijednosti malondialdehida i proteinskih karbonila u potpuno neorganiziranom tumorskom tkivu i teratomskoj liniji s onima u listu hrena, uočena je pojava oksidacijskog stresa iako je izmjeren manji udjel H2O2. Aktivnost lipoksigenaze povećana je u teratomskoj liniji, a još više u potpuno neorganiziranom tumorskom tkivu, što pokazuje da bi peroksidacija masnih kiselina ovisna o aktivnosti lipoksigenaze mogla biti jedan od uzroka oksidacijskoga stresa. Štoviše, u transformiranim je tkivima za razliku od lista uočena bitno veća aktivnost peroksidaze i askorbat peroksidaze te veći broj njihovih izoenzima. S druge strane, izmjerena je kudikamo manja aktivnost superoksid dismutaze, s obzirom na smanjeni udjel H2O2. Veća aktivnost katalaze u listu i teratomskoj liniji povezana je s ulogom katalaze u rastu i diferencijaciji. Dobiveni rezultati potvrđuju prisutnost oksidacijskog stresa u dediferenciranim i hiperhidriranim tumorskim tkivima hrena, odnosno da je oksidacijski stres posljedica nesposobnosti teratomske linije da regenerira biljke normalne morfologije