Effects of pH, light and temperature on (1→3,1→4)-β-glucanase stability in wheat leaves

Changes in (1→3,1→4)-β-D-glucan endohydrolase (EC 3.2.1.73) protein levels were investigated in segments from second leaves of wheat (Triticum aestivum L.). The abundance of the enzyme protein markedly increased when leaf segments were incubated in the dark whereas the enzyme rapidly disappeared when dark-incubated segments were illuminated or fed with sucrose. Addition of cycloheximide (CHI) to the incubation medium led to the disappearance of previously synthesized (1→3,1→4)-β-glucanase and suppressed the dark-induced accumulation indicating that the enzyme was rather unstable. The degradation of (1→3,1→4)-β-glucanase was analyzed without the interference of de-novo synthesis in intercellular washing fluid (IWF). The loss of the enzyme protein during incubation of IWF (containing naturally present peptide hydrolases) indicated that the stability increased from pH 4 to pH 7 and that an increase in the temperature from 25 to 35 °C considerably decreased the stability. Chelating divalent cations in the IWF with o-phenanthroline also resulted in a lowered stability of the enzyme. A strong temperature effect in the range from 25 to 35 °C was also observed in wheat leaf segments. Diurnal changes in (1→3,1→4)-β-glucanase activity were followed in intact second leaves from young wheat plants. At the end of the dark period, the activity was high but constantly decreased during the light phase and remained low if the light period was extended. Activity returned to the initial level during a 10-h dark phase. During a diurnal cycle, changes in (1→3,1→4)-β-glucanase activity were associated with reciprocal changes in soluble carbohydrates. The results suggest that the synthesis and the proteolytic degradation of an apoplastic enzyme may rapidly respond to changing environmental conditions.

Clonal Evolution and Blast Crisis Correlate with Enhanced Proteolytic Activity of Separase in BCR-ABL b3a2 Fusion Type CML under Imatinib Therapy.

Unbalanced (major route) additional cytogenetic aberrations (ACA) at diagnosis of chronic myeloid leukemia (CML) indicate an increased risk of progression and shorter survival. Moreover, newly arising ACA under imatinib treatment and clonal evolution are considered features of acceleration and define failure of therapy according to the European LeukemiaNet (ELN) recommendations. On the basis of 1151 Philadelphia chromosome positive chronic phase patients of the randomized CML-study IV, we examined the incidence of newly arising ACA under imatinib treatment with regard to the p210BCR-ABL breakpoint variants b2a2 and b3a2. We found a preferential acquisition of unbalanced ACA in patients with b3a2 vs. b2a2 fusion type (ratio: 6.3 vs. 1.6, p = 0.0246) concurring with a faster progress to blast crisis for b3a2 patients (p = 0.0124). ESPL1/Separase, a cysteine endopeptidase, is a key player in chromosomal segregation during mitosis. Separase overexpression and/or hyperactivity has been reported from a wide range of cancers and cause defective mitotic spindles, chromosome missegregation and aneuploidy. We investigated the influence of p210BCR-ABL breakpoint variants and imatinib treatment on expression and proteolytic activity of Separase as measured with a specific fluorogenic assay on CML cell lines (b2a2: KCL-22, BV-173; b3a2: K562, LAMA-84). Despite a drop in Separase protein levels an up to 5.4-fold increase of Separase activity under imatinib treatment was observed exclusively in b3a2 but not in b2a2 cell lines. Mimicking the influence of imatinib on BV-173 and LAMA-84 cells by ESPL1 silencing stimulated Separase proteolytic activity in both b3a2 and b2a2 cell lines. Our data suggest the existence of a fusion type-related feedback mechanism that posttranslationally stimulates Separase proteolytic activity after therapy-induced decreases in Separase protein levels. This could render b3a2 CML cells more prone to aneuploidy and clonal evolution than b2a2 progenitors and may therefore explain the cytogenetic results of CML patients.

Light intensity selectively influences the distribution and further redistribution of macro- and micronutrients in hydroponically grown wheat ( Triticum aestivum L.)

Investigations were focused on light effects on allocation of root-borne macronutrients (calcium, magnesium and potassium) and micronutrients (iron, manganese, zinc and copper) in roots, shoots and harvested grains of wheat (Triticum aestivum L.). Plants were exposed to low (100 μmol photons m−2 s−1) or high light (380 μmol photons m−2 s−1). High light stimulated both root and shoot growth. While the total contents per plant of some nutrients were markedly higher (calcium and potassium) or lower (copper) under high light, no major differences were observed for other nutrients. The distribution of nutrients and the further redistribution within the shoot were influenced by the light intensity in an element-specific manner. Nutrients were selectively directed to the leaves of the main shoot (low light) or to the tillers (high light). The quality of the harvested grains was also affected by the light intensity.

Reversible accumulation of (1→3,1→4)‐β‐glucan endohydrolase in wheat leaves under sugar depletion

A (1→3,1→4)‐β‐D‐glucan endohydrolase [(1→3,1→4)‐β‐glucanase, EC 3.2.1.73] was detected in wheat (Triticum aestivum L.) leaves by Western analyses and activity measurements. This enzyme is able to degrade the (1→3,1→4)‐β‐glucans present in the cell walls of cereals and other grass species. In wheat, enzyme levels clearly increased during leaf development, reaching maximum values at full expansion and then decreasing upon leaf ageing. To test whether the abundance of (1→3,1→4)‐β‐glucanase might be controlled by the carbohydrate status, environmental and nutritional conditions capable of altering the leaf soluble sugar contents were used. Both the activity and enzyme protein levels rapidly and markedly increased when mature leaves were depleted of sugars (e.g. during extended dark periods), whereas elevated carbohydrate contents (e.g. following continuous illumination, glucose supply in the dark or nitrogen deficiency during a light/dark cycle) caused a rapid decrease in (1→3,1→4)‐β‐glucanase abundance or prevented its accumulation in the leaves. The physiological significance of (1→3,1→4)‐β‐glucanase accumulation under sugar depletion remains to be elucidated.

Transfer of Cesium from the Xylem to the Phloem in the Stem of Wheat

Steam-girdling experiments with detached wheat shoots showed that cesium was eliminated from the xylem sap and loaded into the phloem during acropetal transport. This transfer is important for the accumulation of cesium (especially also of the radiopollutants 134Cs and 137Cs) in maturing wheat grains.

Long-distance transport of alkali metals in maturing wheat

The alkali metals cesium, rubidium, lithium and sodium were introduced together with strontium via flaps into leaf laminas or into the stem of maturing, intact winter wheat (Triticum aestivum L. cv. Arina) grown in a field. Long-distance transport of these elements and the influence of the application date and of different application positions were investigated. The phloem-immobile Sr served as a marker for the distribution of the xylem sap in the plants. Dry matter accumulation in the grains and the transpiration per shoot were not markedly affected by the treatments as compared to control plants. The phloem mobility was rather high for Cs and Rb. Li was almost immobile in the phloem (similarly to Sr). An application into the cut stem xylem below the second leaf node contributed more to the contents in the grains than an application into the flag leaf. An earlier feeding date led to a higher accumulation in the grains. The marked losses of the elements applied during maturation (most pronounced for Li) can be explained by leakage in the rain.

CO2, light and temperature influence senescence and protein degradation in wheat leaf segments

Effects of environmental conditions influencing photosynthesis and photorespiration on senescence and net protein degradation were investigated in segments from the first leaf of young wheat (Triticum aestivum L. cv. Arina) plants. The segments were floated on H2O at 25, 30 or 35°C in continuous light (PAR: 50 or 150 µmol m−2 s−1) in ambient air and in CO2-depleted air. Stromal enzymes, including phosphoglycolate phosphatase, glutamine synthetase, ferredoxin-dependent glutamate synthase, phosphoribulokinase, and the peroxisomal enzyme, glycolate oxidase, were detected by SDS-PAGE followed by immunoblotting with specific antibodies. In general, the net degradation of proteins and chlorophylls was delayed in CO2-depleted air. However, little effect of CO2 on protein degradation was observed at 25°C under the lower level of irradiance. The senescence retardation by the removal of CO2 was most pronounced at 30°C and at the higher irradiance. The stromal enzymes declined in a coordinated manner. Immunoreactive fragments from the degraded polypeptides were in most cases not detectable. However, an insolubilized fragment of glycolate oxidase accumulated in vivo, especially at 25°C in the presence of CO2. Detection of this fragment was minimal after incubation at 30°C and completely absent on blots from segments kept at 35°C. In CO2-depleted air, the fragment was only weakly detectable after incubation at 25°C. The results from these investigations indicate that environmental conditions that influence photosynthesis may interfere with senescence and protein catabolism in wheat leaves.

Redistribution of cobalt and nickel in detached wheat shoots: effects of steam-girdling and of cobalt and nickel supply

Detached wheat shoots (ear with peduncle and flag leaf) were incubated for 4 d in a solution containing 1 mM RbCl and 1 mM SrCl2 as well as 10, 40 or 160 µM NiCl2 and CoCl2. The phloem of some plants was interrupted by steam-girdling the stem below the ear to distinguish between xylem and phloem transport. The phloem-immobile Sr flowed mainly to the leaf lamina and to the glumes via the xylem. The Sr transport was not sensitive to steam-girdling. In contrast, the phloem-mobile Rb accumulated during the incubation time mainly in the stem and the leaf sheath. The Rb transport to the grains was impaired by steam-girdling as well as by elevated Ni and Co concentrations in the incubation solution indicating that Rb was transported via the phloem to the maturing grains and that this transport was affected by the heavy metals. Ni was removed more efficiently from the xylem in the peduncle than Co (but far less efficiently than Rb). It became evident that the two heavy metals can also be transferred from the xylem to the phloem in the stem of wheat and reach the maturing grains via the phloem.

Long-distance transport of cobalt and nickel in maturing wheat

Cobalt, nickel and strontium were introduced via flaps into leaf laminas or into the stem of maturing, intact winter wheat (Triticum aestivum L., cv. `Arina') grown under natural conditions in a field. Long-distance transport of these elements and the influence of the application date and of different application positions were investigated. The dry-matter accumulation in the grains was not markedly affected by the treatments as compared to untreated control plants. The phloem-immobile strontium served as a marker for the distribution of the xylem sap in the plants. After foliar application, nickel accumulated more rapidly and in higher quantities in the grains than cobalt. Therefore, nickel has a slightly better phloem mobility than cobalt. Regardless of the application date, a higher percentage of the two elements was transported from the flag leaf lamina than from the second or third lamina from the top to the grains. These results indicate that the leaf position is highly relevant for the transfer of the heavy metals investigated to the ear. Introduction into the stem led to a higher accumulation of nickel and cobalt in the grains than introduction into one of the leaves. An earlier feeding date caused a higher accumulation of nickel and cobalt in the grains when introduced into the stem. In contrast, no major differences between earlier and later feeding dates were detected when the elements were introduced into the leaves. Losses of the applied elements were detected during maturation and can be explained by leakage in the rain.

The Chilean wheat market and its price support mechanism : a spatial market integration analysis

This research investigates the spatial market integration of the Chilean wheat market in relation with its most representative international markets by using a vector error correction model (VECM) and how a price support policy, as a price band, affect it. The international market was characterized by two relevant wheat prices: PAN from Argentina and Hard Red Winter from the United States. The spatial market integration level, expressed in the error correction term (ECT), allowed concluding that there is a high integration degree among these markets with a variable influence of the price band mechanism mainly related with its estimation methodology. Moreover, this paper showed that Chile can be seen as price taker as long as the speed of its adjustment to international shocks, being these reactions faster than in the United States and Argentina. Finally, the results validated the "Law of the One Price", which assumes price equalization across all local markets in the long run.

Analysis of duplication in the Spanish durum wheat collection maintained in the CRF-INIA on the basis of agro-morphological traits and gliadin proteins

A total of 106 potential duplicate cases involved 277 accessions were detected on the basis of passport data in the durum wheat collection maintained in the CRF-INIA. Similarity between accessions was measured by agro-morphological traits. The 90% of the agro-morphological duplication were verified with gliadin proteins, allowing identification of similar material with greater refinement than agro-morphological data. However, the results indicated not to decide for rationalisation only on the basis of molecular data.

Analysis of duplication in the Spanish durum wheat collection maintained in the CRF-INIA on the basis of agro-morphological traits and gliadin proteins

A total of 106 potential duplicate cases involved 277 accessions were detected on the basis of passport data in the durum wheat collection maintained in the CRF-INIA. Similarity between accessions was measured by agro-morphological traits. The 90% of the agro-morphological duplication were verified with gliadin proteins, allowing identification of similar material with greater refinement than agro-morphological data. However, the results indicated not to decide for rationalisation only on the basis of molecular data

Combined use of gliadins and SSRs to analyse the genetic variability of the Spanish collection of cultivated diploid wheat (Triticum monococcum L. ssp. monococcum)

This work studied the combined use of gliadins and SSRs to analyse inter- and intra-accession variability of the Spanish collection of cultivated einkorn (Triticum monococcum L. ssp. monococcum) maintained at the CRF-INIA. In general, gliadin loci presented higher discrimination power than SSRs, reflecting the high variability of the gliadins. The loci on chromosome 6A were the most polymorphic with similar PIC values for both marker systems, showing that these markers are very useful for genetic variability studies in wheat. The gliadin results indicated that the Spanish einkorn collection possessed high genetic diversity, being the differentiation large between varieties and small within them. Some associations between gliadin alleles and geographical and agro-morphological data were found. Agro-morphological relations were also observed in the clusters of the SSRs dendrogram. A high concordance was found between gliadins and SSRs for genotype identification. In addition, both systems provide complementary information to resolve the different cases of intra-accession variability not detected at the agro-morphological level, and to identify separately all the genotypes analysed. The combined use of both genetic markers is an excellent tool for genetic resource evaluation in addition to agro-morphological evaluation.