Asuman Cansev

Cold-hardiness of olive (Olea europaea L.) cultivars in cold-acclimated and non-acclimated stages: seasonal alteration of antioxidative enzymes and dehydrin-like proteins.

SUMMARY Seasonal patterns of antioxidative enzymes and proteins and their relations to cold-hardiness of nine olive (Olea europaea L.) cultivars (Ascolona, Domat, Gemlik, Hojoblanca, Lecquest, Manzanilla, Meski, Samanli and Uslu) are documented in the current study. Fully expanded, uniformly sized leaves from 2-year-old shoots of the cultivars were collected from 20-year-old trees in cold-acclimated (CA, in January) and non-acclimated (NA, in July) stages. Leaf samples were exposed to low temperature at 4, x5, x10 and x20 xC for 12 h to determine their cold-hardiness (LT50; assessed by electrolyte leakage). Cold-acclimation produced an increase in freezing tolerance of all cultivars (by lowering LT50). Domat and Lecquest were found to have the highest cold-hardiness among the nine cultivars investigated. Ascolona, Gemlik, Hojoblanca had moderate cold-hardiness, while Samanli, Meski, Uslu and Manzanilla were more sensitive. Activities of catalase (CAT : EC 1 . 11 . 1 . 6), ascorbate peroxidase (APX : EC 1 . 11 . 1 . 11) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase significantly varied depending on the cold-acclimation stage and the cold-hardiness level of the cultivars. Activities of the three antioxidative enzymes and total soluble proteins (TSP) were higher in the CA stage than in the NA stage. Although no accumulation of major polypeptides, except a 23 kDa protein, was detected either in CA samples or NA samples by SDS-PAGE, anti-dehydrin immunoblots revealed that the 43 and 23 kDa polypeptides were detectable during cold-acclimation of olive cultivars. Accumulation of both 43 and 23 kDa dehydrin was significantly higher in the CA stage than in the NA stage in all cultivars. Accumulation of 43 kDa dehydrin was correlated with cold-hardiness of the cultivars, while 23 kDa dehydrin was considered as cultivar-dependent since its accumulation was not parallel to LT50 values of the cultivars. Indeed, the tissues of cvs Domat, Lecquest, Ascolona, Hojoblanca and Gemlik were found to enhance the structural stability of cellular membranes in the CA stage by increasing both the activity of such enzymes as CAT, APX and NADPH oxidase to activate the antioxidative systems and the expression of 43 kDa dehydrins.

Annual patterns of total soluble sugars and proteins related to coldhardiness in olive (Olea europaea L.‘Gemlik’)

Summary Annual patterns of cold hardiness, total soluble sugars (TSS) and proteins (TSP) were characterised during two consecutive years (May 2003–April 2005) in leaf and bark tissues of olive (Olea europaea L.) ‘Gemlik’ trees. One-year-old shoots from 13-year-old olive trees were collected each month and exposed to low temperature at 4°C, –5°C, –10°C, or –20°C for 12 h to determine their cold-hardiness (LT50; assessed by electrolyte leakage). A considerable increase in cold-hardiness was observed with decreasing temperature. Leaf and bark tissues exhibited an increase in cold-hardiness during the Autumn, which reached a maximum in mid-Winter, then gradually decreased during the Spring, and reached a minimum in mid-Summer. Results indicated that leaf and bark tissues in both experimental years responded similarly to cold, with a continuous increase in their TSS and TSP contents during field adaptation in Autumn and Winter, which paralleled their cold-hardiness. The annual cycle of TSP from leaf and bark tissue was characterised by one dimensional SDS-PAGE. A seasonal fluctuation was observed in leaf proteins of 66 kDa and 43 kDa, and in bark proteins of 70 kDa, 43 kDa and 16 kDa, which paralleled cold-acclimation. Evaluation of these leaf and bark TSP profiles showed that these polypeptides disappeared in the Summer, followed by their accumulation again during the Autumn and Winter. Moreover, all these polypeptides became less visible during the Spring. The relationships between these five proteins, TSS contents, and cold-hardiness in olive are discussed.

The activities of catalase and ascorbate peroxidase in olive (Olea europaea L. cv. Gemlik) under low temperature stress

In this study, one-year-old shoots of the olive (Olea europaea L.) cv. Gemlik were tested at artificial low temperatures (4, −5°C, −10°C, and −20°C) every month for two years. For low temperature treatment, the degree of cell membrane injury in leaves and barks was determined by ion leakage method. In addition, with regard to antioxidative defense mechanism, activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) enzymes were determined. Leaf and bark tissues subjected to 4°C and −5°C injured to a limited extent in all months. However, more than 50% injury occurred by temperatures equal to or colder than −10°C treatments depending on the season. For −10°C and −20°C treatments, the lowest and the highest injury in leaf and bark tissues were detected during winter and summer seasons, respectively. We determined in this study that CAT and APX enzyme activities are generally higher during fall and winter compared with those in summer. On the other hand, CAT and APX enzyme activities started increasing during fall along with a decreasing freezing injury while the activities of these enzymes decreased to some extent during winter when freezing injury was the lowest. In addition, while CAT activity decreased with low temperature treatments, APX activity did not change until −5°C treatment but decreased with decreasing temperatures starting from −10°C depending on the month the tissue was obtained. In conclusion, olive plant shows considerable tolerance to low temperatures that are achieved after daily gradual decreases by increasing cell membrane stability through complicated mechanisms including antioxidative enzyme metabolisms. In addition, APX may be more effective in maintaining cold-hardiness of olive compared with CAT.

Cold hardiness of olive (Olea europaea L.) cultivars in cold-acclimated and non-acclimated stages: seasonal alteration of soluble sugars and phospholipids.

In many plant species, several physiological and biochemical changes occur during low-temperature-induced cold acclimation. A previous study with olive cultivars (Cansev et al. 2009) demonstrated a correlation between the level of accumulation of certain leaf proteins besides antioxidative enzyme activities and cold hardiness of the cultivars. The present paper analysed soluble sugar (SS) and phospholipid (PL) contents of cold-acclimated (CA) and non-acclimated (NA) leaf tissues in order to explain the mechanism of cultivar-dependent response to cold in olive. In general, cold acclimation significantly increased total soluble sugar (TSS), reducing sugars and sucrose contents of all cultivars to various extents depending on the cold hardiness of cultivars. In addition, TSS, reducing sugars and sucrose contents in cold-tolerant cultivars were significantly increased, whereas TSS, reducing sugars and sucrose contents in cold-sensitive cultivars either did not change or increased slightly in CA stage compared with those in NA stage. Even though reducing sugars were the major soluble sugar in olive leaves, levels of sucrose accumulations in CA stage compared with those in NA stage were greater than those observed in reducing sugars accumulation. Changes in levels of total PL, as well as the three individual PL fractions phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), were investigated in olive leaf tissues. Significant increases in levels of PC and PE fractions during CA compared with those in NA stage suggested that PC and PE maintained the cold hardiness of olive cultivars more effectively than did PI. Although the precise mechanisms by which olive responds to cold may still be open to discussion, soluble sugars and PL are clearly important in the ability of olive cultivars to stand against cold stress.

Development and validation of new SSR markers from expressed regions in the garlic genome

Only a limited number of simple sequence repeat (SSR) markers is available for the genome of garlic (Allium sativum L.) despite the fact that SSR markers have become one of the most preferred DNA marker systems. To develop new SSR markers for the garlic genome, garlic expressed sequence tags (ESTs) at the publicly available GarlicEST database were screened for SSR motifs and a total of 132 SSR motifs were identified. Primer pairs were designed for 50 SSR motifs and 24 of these primer pairs were selected as SSR markers based on their consistent amplification patterns and polymorphisms. In addition, two SSR markers were developed from the sequences of garlic cDNA-AFLP fragments. The use of 26 EST-SSR markers for the assessment of genetic relationship was tested using 31 garlic genotypes. Twenty six EST-SSR markers amplified 130 polymorphic DNA fragments and the number of polymorphic alleles per SSR marker ranged from 2 to 13 with an average of 5 alleles. Observed heterozygosity and polymorphism information content (PIC) of the SSR markers were between 0.23 and 0.88, and 0.20 and 0.87, respectively. Twenty one out of the 31 garlic genotypes were analyzed in a previous study using AFLP markers and the garlic genotypes clustered together with AFLP markers were also grouped together with EST-SSR markers demonstrating high concordance between AFLP and EST-SSR marker systems and possible immediate application of EST-SSR markers for fingerprinting of garlic clones. EST-SSR markers could be used in genetic studies such as genetic mapping, association mapping, genetic diversity and comparison of the genomes of Allium species.

Effect of processing techniques on antioxidative enzyme activities, antioxidant capacity, phenolic compounds, and fatty acids of table olives

In this study, olive fruits (Olea europeae cv. Gemlik) of the most common sources of table olives in Turkey were used. Total polyphenol content (TPC), antioxidant capacity (AC), and antioxidant enzymes (catalase, CAT; ascorbate peroxidase, APX; and glutathione reductase, GR) of table olives were compared by 4 different methods of ripe table olive processing. Results revealed that TPC of the processed olives ranged from 117.44 to 418.69 mg gallic acid equivalents/g fresh weight (f.w.). The highest AC as mg Trolox equivalents of 189.58/g f.w. was obtained from unprocessed black olives. CAT, APX, and GR activities of unprocessed olives were higher than those obtained in all processed olives. In conclusion, TPC, AC, and antioxidant enzyme activities are strongly affected by fruit ripening and processing in table olives of ‘Gemlik’ cultivar. In addition, the best processing technique is untreated black olives in brine for antioxidant properties.

Chemical and techno-functional properties of flours from peeled and unpeeled oleaster ( Elaeagnus angustifolia L.)

Oleaster flours were produced from two different genotypes (GO1 and GO2) and methods (peeled oleaster flour: POF and unpeeled oleaster flour: UPOF). Oleaster flour samples (OFs) contained high levels of dietary fibers and micro minerals. The contents of Fe, Cu, B, and Cr in flours obtained from oleaster fruits were higher in UPOF than in POF samples. Palmitic acid was the major fatty acid which was followed by oleic acid and lignoceric acid. All samples contained greater amount of saturated fatty acids (SFA) as compared to mono unsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). Among seven different organic acids detected, the level of citric acid was the highest and it was followed by malic, acetic and oxalic acids. High nutritional contents of oleaster flour indicated that it is a good source of dietary fiber, micro minerals, as well as organic and fatty acids. The water solubilities (WS) and water absorption capacities (WAC) of oleaster flours were adequate for their utilization. They also seem to have an improving effect on emulsion properties of albumin. These results highlighted that it is possible to use the oleaster flour in some processed foods such as bakery goods, dairy products (ice cream and yoghurt), beverages and confectionery. Moreover, the oleaster flour could also be used in the preparation of low-fat, high-fiber dietetic products due to its high dietary fiber content.