Purification and partial characteristic of a major gliadin-degrading cysteine endopeptidase from germinating triticale seedsPrabucka, Beata; Bielawski, Wiesław
doi: 10.1007/s11738-004-0027-6pmid: N/A
The endopeptidase of the highest electrophoretic mobility was the main endopeptidase hydrolyzing gliadin in the endosperm of germinated triticale (X Triticosecale Wittm.) grains after three days of imbibition. Activity of this endopeptidase, named EP8 starts to be detectable after two days of imbibition. The appearance of its activity in the endosperm on a second day of imbibition may suggest that EP8 is synthesized in aleurone during germination and/or secreted into the starchy endosperm as an inactive polypeptide during grains development and then activated. EP8 was isolated from the endosperm of germinating triticale seeds and purified 257-fold using ammonium sulphate, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-100. The enzyme was totally inhibited by E-64—class-specific cysteine proteinases inhibitor and activated by thiol compounds. Molecular weight estimated by SDS-PAGE was 39.5 kDa. The optimum pH for the hydrolysis of gliadin was 4.2 and for hemoglobin 5.2. High activity of EP8 against wheat gliadin in vitro suggests that this cysteine endopeptidase plays a major role in the mobilization of storage proteins in the endosperm of germinating triticale grains.
A new acylated flavonol diglycoside from Atriplex littoralisBylka, Wiesława
doi: 10.1007/s11738-004-0028-5pmid: N/A
A new acetylated flavonol glycoside: patuletin 3-O-[5′″-O-feruloyl-β-D-apiofuransyl (1′″→2′′)-β-D-glucopyranoside] (2), together with a known patuletin 3-O-β-D-glucopyranoside (1) were isolated from the aerial part of Artiplex littoralis L. (Chenopodiacease). Their structures were elcidated by acid hydrolysis and spectroscopic methods including UV, 1H, 13C NMR and ESI-MS for both compounds, additionally 2D-NMR, HSQC, HMBC experiments were performed for 2.
Ammonium and proline accumulation in senescing cut leaves of ZantedeschiaRabiza- wider, Julita; Łukaszewska, Aleksandra; Skutnik, Ewa; Leszko, Magdalena
doi: 10.1007/s11738-004-0032-9pmid: N/A
Accumulation of ammonium and proline were reported as phenomena associated with plant response to stress and/or senescence. The effects of a preservative (8HQC + sucrose) and 24 hrs pulse conditioning with GA3 on the ammonium and proline contents were studied in senescing cut leaves of Zantedeschia aethiopica Spr. and Z. elliottiana Engl., grown for the florists green. Generally, accumulation of both compounds was observed in senescing leaves, however, the final ammonium and proline levels depended upon the species and the treatment applied. Conditioning with GA3, a treatment known to delay leaf senescence in Zantedeschia sp., prevented the increases in the ammonium and proline contents. Standard preservative solution used to prolong the longevity of cut flowers enhanced the ammonium accumulation in senescing leaves of both species, and the proline accumulation in the leaves of Z. aethiopica, but not in Z. elliottiana. These observations suggest that neither ammonium nor proline accumulation would be fully reliable predictors of cut leaf freshness during their entire market life. However, proline accumulation could serve as a quick test of freshness in the first half of the useful market life of cut leaves of Zantedeschia.
UV-B and cadmium induced changes in pigments, photosynthetic electron transport activity, antioxidant levels and antioxidative enzyme activities of Riccia sp.Prasad, Sheo; Dwivedi, Rajiv; Zeeshan, Mohd.; Singh, Ranjana
doi: 10.1007/s11738-004-0033-8pmid: N/A
UV-B and cadmium, alone and together, induced changes in photosynthetic pigment levels, photosynthetic electron transport activity, enzymatic and non-enzymatic (low molecular weight) antioxidants, level of hydrogen peroxide and lipid peroxidation in Riccia sp. were evaluated. Chlorophyll content was found to decrease with the rising concentration of cadmium and UV-B exposure alone and its level further declined when both the stresses were applied together. In contrast to this, carotenoids exhibited varied response, as it showed enhancement with UV-B (15, 30 and 45 min exposure) and low concentration of Cd (1 and 10 µM) treatment alone and in combination. Both the stresses caused strong inhibitory effect on PS II activity (H2O → p-BQ), while PS I activity (DCPIP/ASC → MV) appeared to be less sensitive. Total peroxide content increased with simultaneous increase in lipid peroxidation. The level of non-enzymatic antioxidant ascorbate and enzymatic antioxidants superoxide dismutase and peroxidase activity were found to increase with simultaneous decrease in catalase activity following UV-B and Cd treatments. These results indicate that 45 min of UV-B exposure and 10, 100 and 1000 µM cadmium alone and together, strongly arrested electron flow through PS II which caused accelerated generation of reactive oxygen species (H2O2) and excess accumulation of H2O2 due to significant inhibition of catalase activity, led to the oxidative damage in Riccia sp.
Effect of water deficit on oxidative stress and degradation of cell membranes in needles of Norway spruce (Picea abies)Kami ska-Ro ek, Emilia; Pukacki, Paweł
doi: 10.1007/s11738-004-0034-7pmid: N/A
We studied the impact of mild and severe drought stresses for 42 days and rehydration for 21 days on 4-year-old seedlings of Norway spruce. Water relations in spruce tissues were determined on the basis relative water content of needles and shoot water potential (Ψshoot). During the stress, we measured the level of: reactive oxygen species (ROS), antioxidants, and degradation of cell membranes. In the seedlings subjected to severe stress, Ψshoot decreased to −2.4 MPa, while in those subjected to mild stress, to −0.8 MPa. After rehydration, shoot water potential increased, but did not reach the control level. Water deficit caused oxidative stress, reflected in an increased production of ROS: superoxide anion radical (
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) and hydrogen peroxide (H2O2). Their concentrations in needles were the highest in seedlings subjected to severe stress, where they exceeded the control level by 116% and 30%, respectively. During rehydration, the differences in ROS levels between treated and control seedlings diminished. Oxidative stress causing degradation of cell membranes included: de-esterification of phospholipids, oxidation of fatty acids, and increase in concentration of malondialdehyde, as their permeability to ions increased by 125%. In the defence against the oxidative stress in needles, an important role was played by low-molecule antioxidants such as glutathione, ascorbic acid, flavonoids, α-tocopherol and antioxidant enzymes. An increase in intensity of water deficit caused a significant reductio in the level of low-molecular antioxidants, which attests to their utilization during the process of scavenging for free radicals. Water deficit at Ψshoot=−1.7 MPa caused a decline in ascorbic acid level by 37% in needle cells. An effective defensive mechanism removing the excess of ROS was also reflected in the activity of the main enzymes of oxidative stress: superoxide dismutase (SOD) and guaiacol peroxidase (PO). As a result of water deficit, SOD activity increased by 80 %, while PO activity decreased by 82 %.
Variation of total soluble seminal root proteins of tetraploid wild and cultivated wheat induced at cold acclimation and freezingTerzioglu, Serpil; Ekmekci, Yasemin
doi: 10.1007/s11738-004-0035-6pmid: N/A
The relationship between total soluble seminal root proteins induced at cold acclimation and freezing tolerance in tetraploid wild wheat Aegilops L. (Ae. biuncialis, Ae. cylindrica) and cultivated wheat Triticum turgitum L. (Firat-93, Harran-95) was investigated. Cold acclimation was performed at 0 °C for 7 days. Freezing tolerance was determined with survived roots after freezing treatments at −5 and/or −7 °C for 3, 6, 12 and 24 h. At −5°C, all tetraploid genotypes showed over 60% tolerance for 3 h. This effect was also present in wild wheat for 6 h, but was decreased in cultivated wheat to 30–35% tolerance for 6 h. Only Ae. biuncialis was able to show 52% tolerance just for 3 h freezing period at −7 °C. However, all the genotypes were not survived at −7 °C, for 6, 12 and 24 h. Cold acclimation induced greater amounts of new soluble seminal root proteins in tolerant Ae. biuncialis (29–104 kDa, pI 5.4–7.4) than in sensitive Harran-95 (29–66 kDa, pI 6.1–8.3). Synthesis and accumulation of these proteins may be related to degree of freezing tolerance of these genotypes.
Infiltration with Agrobacterium — the method for stable transformation avoiding tissue cultureGrabowska, Agnieszka; Filipecki, Marcin
doi: 10.1007/s11738-004-0036-5pmid: N/A
A number of in planta transformation protocols that avoid long culture under sterile conditions were developed for Arabidopsis thaliana. The most widely used methods are based on vacuum infiltration and floral dip. These methods were adapted for transformation of other species as well. Successful in planta transformations of alfalfa, radish, pakchoi and petunia were reported recently. In this short review we present several modified procedures originally developed for Arabidopsis thaliana and in some cases adapted to other species. We emphasize the crucial parameters involved in in planta transformation. We also describe here the studies attempting to shed light on the mechanisms and estimating the cellular target of transformation, which may help in transforming new plant species.