Reena Grittle Pinhero

Changes in Activities of Antioxidant Enzymes and Their Relationship to Genetic and Paclobutrazol-Induced Chilling Tolerance of Maize Seedlings

The potential role of antioxidant enzymes in protecting maize (Zea mays L.) seedlings from chilling injury was examined by analyzing enzyme activities and isozyme profiles of chilling-susceptible (CO 316) and chilling-tolerant (CO 328) inbreds. Leaf superoxide dismutase (SOD) activity in CO 316 was nearly one-half that of CO 328, in which the high activity was maintained during the chilling and postchilling periods. Activity of glutathione reductase (GR) was much higher in roots than in leaves. CO 328 also possessed a new GR isozyme that was absent in roots of CO 316. Ascorbate peroxidase (APX) activity was considerably lower in leaves of CO 328 than in CO 316, and nearly similar in roots. Paclobutrazol treatment of CO 316 induced several changes in the antioxidant enzyme profiles and enhanced their activities, especially those of SOD and APX, along with the induction of chilling tolerance. These results suggest that increased activities of SOD in leaves and GR in roots of CO 328, as well as SOD and APX in leaves and roots of paclobutrazol-treated CO 316, contribute to their enhanced chilling tolerance.

Inhibition of phospholipase D

Phospholipases A2 are involved in inflammatory processes such as the liberation of free arachidonic acid from the membrane pool for the biosynthesis of eicosanoids. Inhibitors of these enzymes are proving useful in determining the biological roles of phospholipases A2 in complex cellular processes and may also have therapeutic potential. Inhibition of these lipolytic enzymes is more difficult to characterize as the enzymatic reaction occurs at a lipid/water interface. This review focuses on the description of a number of classes of rationally designed phospholipase A2 inhibitors. The development of a theoretical framework for the proper analysis of inhibitors is presented. Structural studies of phospholipase A2‐inhibitor complexes suggest how the lipolysis reaction is catalyzed. Finally, some recent results on the use of phospholipase A2 inhibitors in living cells and tissues are revealed.— Gelb, M. H., Jain, M. K., Berg, O. G. Inhibition of phospholipase A2. FASEB J. 8: 916‐924; 1994.

Modulation of phospholipase D and lipoxygenase activities during chilling. Relation to chilling tolerance of maize seedlings

Phospholipase D (phosphatidylcholine choline hydrolase, EC 3.1.4.4) and lipoxygenase activities (linoleate: oxygen oxidoreductase, EC 1.13.11.12) were analysed in chilling-susceptible and chilling-tolerant maize (Zea mays L.) seedlings in relation to their chilling tolerance. Before chilling, phospholipase D activity in the leaf microsomal fraction was 2-fold higher in the chilling-susceptible CO 316 when compared to that of paclobutrazol-treated, chilling-tolerant CO 316. Phospholipase D activity in the heavy membrane fraction comprising chloroplast and mitochondria showed a 3 to 4-fold increase during chilling and post-chilling periods, albeit with temporal variations. An increase in cytosolic phospholipase D activity was observed only in CO 316 and CO 328. Also, phospholipase D activity in the heavy membrane and cytosolic fractions of roots increased in CO 316 during chilling and post-chilling periods. Lipoxygenase activity was low in both leaves and roots of CO 328. Paclobutrazol treatment of CO 316 did not appear to affect the level of cytosolic lipoxygenase activity in roots during the early part of chilling and post-chilling, but increased the activity in leaves. The results suggest that during chilling and post-chilling periods, temporal variations in phospholipase D and lipoxygenase activities in both leaves and roots of CO 316, CO 316P and CO 328 could result in differential metabolism of phospholipids. Enhanced degradation coupled with low levels of turnover could lead to development of chilling injury in the tissue.

Paclobutrazol and ancymidol protect corn seedlings from high and low temperature stresses

Hybrid corn (Zea mays L. cv Pioneer 3902) seeds were imbibed either in distilled water (control) or 40 mM KCl solution containing paclobutrazol (P) and ancymidol (A) at 50 and 25 mg 1−1 respectively and air dried. Seedlings were germinated and grown in a controlled environment cabinet with a day/night temperature of 23/18°C and a photoperiod of 16 h with a PPFD of 150 μE m−2s−1. Ten-day-old seedlings were stressed by exposing them either to a high temperature of 51°C for 8 h or a low temperature of 3°C and a photoperiod of 16 h with a PPFD of 640 μE m−2 s−1 for 48 h. Both stresses, decreased chlorophyll and carotenoid levels. A decrease in fluorescence (Fv/Fm ratio) and increase in leakiness indicated lower photosynthetic efficiency and a loss of membrane integrity. These deteriorative symptoms in the control seedlings were ameliorated by the treatment (P+A) and the growth rates of the treated seedlings as measured by height, fresh and dry weights were greater than the control. Based on evidence from previous studies with triazoles, we suggest that the stress protection conferred by P+A may be mediated to some extent by an enhanced activity of free-radical scavenging systems.

ANTIOXIDANT AND CALMODULIN-INHIBITORY ACTIVITIES OF PHENOLIC COMPONENTS IN FRUIT WINES AND ITS BIOTECHNOLOGICAL IMPLICATIONS

Fruits are rich sources of anthocyanins, flavonoids and phenols that are important nutraceutical components. Processed products of various fruits such as wines and juices, also contain these nutraceuticals, and have been proposed to be the primary components that provide health-beneficiary effects to the cardiovascular system. In the present study, we have evaluated the antioxidant and calmodulin-inhibitory effects of three fruit wines and a red grape wine to provide a comparison in their nutraceutical properties. Using in vitro systems that generate superoxide radicals and hydroxyl radicals, all the wines and their partially purified phenolic extracts were demonstrated to possess strong superoxide and hydroxyl radical scavenging properties. On the basis of specific phenolic content, the summer cherry, blackberry and blueberry wines were 30–40% more efficient in superoxide radical scavenging than red grape wine. Similarly, blueberry wine demonstrated considerably high hydroxyl radical scavenging efficiency than the other wines. The flavonoid aglycones such as catechin and naringenin, were as efficient or more in scavenging superoxide radicals, as ascorbate. As well, these compounds also scavenged hydroxyl radicals, as or more effectively than melatonin, a known hydroxyl radical scavenger. All the wines had components that inhibited calcium and calmodulin-promoted phosphodiesterase activity, indicating their potential to interfere with the calcium second messenger function. Blueberry, blackberry and red wine components were more effective in the inhibition of calmodulin-promoted phosphodiesterase than those from summer cherry wine. Active oxygen scavenging or calmodulin-inhibitory properties appear to be associated with a variety of wine components that may provide synergistic action than any single components in them. This study paves way for the potential use of fermentation biotechnology to optimize the enzymatic release and levels of nutritionally important phenolic components in fruit wines.

The Effects of Genetic Transformation of Tomato with Antisense Phospholipase D cDNA on the Quality Characteristics of Fruits and Their Processed Products

Abstract The physico-chemical characteristics of tomato juice and sauce, prepared from transgenic tomato fruits (Lycopersicon esculentum Mill, var. Celebrity) transformed with an antisense phospholipase D (PLD) cDNA construct, were evaluated. The antisense transformation resulted in a 30–40% reduction of PLD activity in ripe fruits. The transgenic fruits were firmer, possessed better red color, and flavor. The juice and sauce preparations from transgenic fruits showed improved red color, lycopene content, Brookfield viscosity, and increased levels of major flavor volatiles such as hexenal. The dry matter and ash contents, as well as the precipitate weight ratio (PPT) of the transgenic fruit products were significantly higher when compared to the products from control fruits. The vitamin C content of the transgenic fruits was also higher as compared to the control fruits. The results suggest that a reduction in PLD activity may lead to increased membrane stability and preservation of membrane compartmentalization that can have positive quality impacts in transgenic fruits and their products.

Post-harvest Storage of Potatoes

Publisher Summary This chapter discusses the various factors that affect the quality of potatoes during post-harvest storage such as maturity stage of crop (early/late), intended use (table stock/processing/seed), preharvest conditions of crop, harvest and handling conditions, health of the crop such as incidence of pests and diseases, biochemical changes, storage preparations and conditions, and management of storage environment. The potato crop can be separated into late- or early-crop based on their maturity at harvest. Maturity is a complex physiological and morphological condition, which is influenced by several factors including respiration, carbohydrate changes, dry matter content, moisture loss, dormancy, and sprouting. The quality of the potato crop that is stored ultimately determines the quality of the stored product. A good storage management cannot enhance quality out of storage if the health of the tubers is compromised during preharvest conditions. Harvesting immature tubers, when the soil conditions are too wet or dry, and during too-warm weather conditions can affect the quality of tubers. Maintenance of post-harvest quality is critical for both growers and processors.

A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His6-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni2+-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004–14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs.

Chloroplast membrane organization in chilling-tolerant and chilling-sensitive maize seedlings

Summary Resistance of maize ( Zea mays L.) seedlings to chilling-induced damage and its potential bearing on chloroplast membrane organization have been investigated using a chilling-sensitive inbred CO 316 and a chilling-tolerant inbred CO 328. Paclobutrazol treatment of the chilling-sensitive CO 316 (CO 316P) induced chilling tolerance, also causing several morphological and physiological changes, and served as another means of exploring the relation between chloroplast membrane changes and chilling tolerance. Chilling treatment [6°C/2°C (day/night)] resulted in a reduction of Fv/Fm ratio, photosynthetic pigment levels and an increase in leakage of electrolytes. Within 24 h of exposure of CO 316 to chilling, the granal and stromal membrane showed extensive vesiculation and disruption of the granal array. By contrast, the granal and stromal organization of chilling-tolerant CO 328 and P-treated CO 316 remained nearly intact. Our studies suggest that paclobutrazol treatment can alter the membrane deterioration and disassembly processes of the thylakoid membranes of chilling-sensitive CO 316 to make them functionally similar to the chloroplast membrane of genetically chilling-tolerant CO 328.

Evaluation of nutritional profiles of starch and dry matter from early potato varieties and its estimated glycemic impact.

To identify healthier potatoes with respect to starch profiles, fourteen early varieties were evaluated for their dietary fiber, total starch, rapidly digestible (RDS), slowly digestible (SDS), and resistant (RS) starch for nutrition and with regard to estimated glycemic index (eGI) and glycemic load (eGL). While all these profiles were highly dependent on the potato variety, eleven out of fourteen varieties were classified as low GL foods (p<0.05). A strong positive correlation was observed with eGI and RDS (r=0.975-1.00, 0.96-1.00 and 0.962-0.997 for uncooked, cooked and retrograded varieties, respectively), whereas a strong negative correlation was observed between eGI and RS (r=-0.985 to -0.998, -0.96 to -1.00 and -0.983 to -0.999 for uncooked, cooked and retrograded varieties respectively, p<0.05). For the cultivars examined, the present study identified RDS and RS as major starch factors contributing to eGI.