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  • Author or Editor: Jiwan P. Palta x
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In recent years evidence has been presented that implicates the role of free (cytosolic) Ca2+ as a major metabolic and developmental controller in plants. Calcium concentrations in the cytoplasm are kept very low under normal conditions (10-6 to 10-8 M). Small changes in the absolute amount of calcium can create a 10- to 100-fold change in the Ca2+ concentration without upsetting the ionic balance of the ceil. This feature makes Ca2+ an excellent candidate as a second messenger. Thus, a stress induced change in the cytosolic Ca2+ could bring a cellular/plant response to stress. This response is thought to be mediated through activation of Ca2+ and/or Ca2+-calmodulin-dependent protein kinases which in turn mediate the activity of various enzymes via phosphorylation. Recent evidences from the impact of salinity, low temperature, high temperature, and biotic stresses support such a role of calcium. Data on the association between stress-induced injury and perturbation of membrane/cytosolic calcium are available. In addition, evidences for the role of calcium in acclimation to stress have been reported. These studies suggest that manipulation of cellular Ca2+ may be one of the approaches we have on hand to bridge the gap between science and technology.

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Recent studies suggest cold-regulated heat-stable proteins mitigate the potential damaging effects of low water activity associated with freezing. A proposed function of these proteins is stabilization of enzymes during exposure of plants to subzero temperatures. To test this hypothesis for tuber-bearing Solanum L. species we determined the quantitative expression of heat-stable proteins, the qualitative changes in dehydrin proteins, and the capacity of heat-stable proteins to cryoprotect a freeze-thaw labile enzyme lactate dehydrogenase (LDH). We used five tuber-bearing Solanum species (S. tuberosum L. `Red Pontiac', S. acaule Bitter, S. sanctae rosea Hawkes, S. commersonii Dunal, and S. cardiophyllum Bitter), which vary in nonacclimated relative freezing tolerance (NA RFT), acclimated relative freezing tolerance (AC RFT), and acclimation capacity (ACC). The protein fraction containing a mixture of heat-stable proteins demonstrated cryoprotective capacities greater or equal to other cryoprotective compounds (bovine serum albumin, polyethylene glycol, glycerol, and sucrose). Heat-stable proteins extracted from acclimated S. commersonii had superior cryoprotective capacity than those extracted from nonacclimated S. commersonii plants. Interestingly, in the presence of these proteins extracted from acclimated plants (in S. commersonii and S. sanctae rosea), LDH activity was elevated above that of unfrozen controls. No quantitative relationships were found between heat-stable protein concentration and NA RFT, AC RFT, or ACC among the five species. This was also true for dehydrin protein expression. Cold acclimation treatment resulted in increased dehydrin expression for acclimating and nonacclimating species. In three of the cold acclimating species (S. acaule, S. sanctae rosea, and S. commersonii), an increase in dehydrin expression may play a role in increased freezing tolerance during cold acclimation. In the cold sensitive, nonacclimating species (S. tuberosum and S. cardiophyllum), however, an increase in dehydrin level maybe related to the response of these species to changed (perhaps stressful) environment during cold treatment. By exploiting the genetic variation in NA RFT and ACC for five tuber-bearing species, we were able to gain new insight into the complexity of the relationship between heat-stable protein and cold response.

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Tuber tissue calcium has been linked to several potato quality characteristics, including internal defects and the susceptibility of tubers to decay by soft rotting Erwinia species. We were particularly interested in studying the relationship between supplemental calcium fertilization during the seed tuber production cycle to raise the seed piece calcium concentration and the impact on crop performance the following season. The role of seed tuber tissue calcium level on seed piece decay, growth, development, and performance of the plant was evaluated for cultivars Russet Burbank, Dark Red Norland, Atlantic, Superior, and Snowden. This study was performed over four growing seasons. Seed tubers were raised with varying calcium and the following season, individual tubers (over 3,000 total for 4 years of study) were sampled for calcium and hand planted in the field. They were evaluated for seed piece decay and total tuber yield during the growing season. Seed tubers raised with supplemental calcium resulted in significantly higher mean calcium content than the control tubers. In general, calcium-raised seed tubers tended to produce a more vigorous main sprout and higher tuber yield. We also found that there are significant differences among these cultivars for the characteristics measured. Consistently, in all three years, `Atlantic' responded to test conditions with the lowest decay values, and `Dark Red Norland' consistently showed the highest decay values. This suggests that there may be a genetic component involved in these two responses and these genotypic differences could be exploited to improve cultivated potatoes.

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High temperature effects potato production by reducing overall growth and partitioning of photosynthate to tubers. Recent studies from our laboratory demonstrated that these effects can be reduced by increasing rhizospheric calcium. This present study was conducted to determine if this mitigation of heat stress effect on potato is due to modulation of heat shock protein by calcium during stress. An inert medium and nutrient delivery system capable of maintaining precise rhizospheric calcium levels were used. Biomass was measured and protein samples were collected from potato leaves. Using electroblotting, heat shock proteins were detected by antibodies to Hsp21 and Hsp70 (obtained from Dr. Elizabeth Vierling). Injury by prolonged heat stress was mitigated at calcium concentration >5 ppm. The calcium concentration of leaf and stem tissues were twice as high in 25 ppm calcium-treated plant compared to 1 ppm calcium-treated plants. Total foliage fresh weight was 33% higher and dry weight 20% higher in plants supplied with 25 ppm of calcium than supplied with 1 ppm of calcium. HSP21 was expressed only at high temperature and at greater concentrations in 25 ppm calcium treatment. HSP70 was expressed in both control, 20 °C/15 °C (day/night) and heat-stressed tissue, 35 °C/25 °C (day/night) under various calcium treatments (1 to 25 ppm). Also, there were some differences in HSPs expression patterns between young and mature leaves. Young tissue responded immediately to the heat stress and started to express HSP21 within 1 day. Mature tissue started to express HSP21 after 2 days. HSP21 of young tissue disappeared sooner than mature tissue when heat stress-treated plants were returned to normal conditions. These results support our earlier studies indicating that an increase in rhizospheric calcium mitigate heat stress effects on the potato plant. Furthermore these results suggest that this mitigation may be due to modulation of HSP21by rhizospheric calcium during heat stress.

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Previous studies in our laboratory both in pine needles and potato leaves have shown evidence of an increase in 18: 2 (linoleate) in the purified plasma membrane fraction during cold acclimation. This increase was reversible on deacclimation, thereby suggesting a link between the accumulation of 18: 2 and acquisition of freezing tolerance. These studies suggest that the activity of specific desaturases may be modulated during cold acclimation. This study was aimed at studying the possible involvement of stearoyl-ACP desaturase (delta9) in potato cold acclimation response. Our approach was to study the induction of delta9 desaturase at the transcript level by using potato delta9 desaturase gene specific primers and reverse transcriptase. For this purpose, mRNA from S. tuberosum (cold sensitive, unable to acclimate) and S. commersonii (cold tolerant, able to cold acclimate) was extracted before and after acclimation. Sequence analysis confirmed that the amplified band was delta9 desaturase. Our results show that there is an increase in delta9 desaturase gene transcripts during cold acclimation and that this increase is associated with the cold acclimation response in potato. These results together with previous reports on the increase in 18: 2 in the plasma membrane during cold acclimation give more evidence toward the involvement of stearoyl-ACP desaturase (delta9) in the potato cold response.

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Lipids have been thought to be important largely in membrane structure and energy reserve. It is now evident that lipids and lipid-derived metabolites play a role in many critical cellular processes. Recent studies have shown that membrane lipid-based signaling mediated by phospholipases such as phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD) constitutes a crucial step in plant responses to abiotic and biotic stresses. Phospholipases and their products also play a role during plant growth and development. For example, PLA2-derived lysophospholipids acted as growth regulators that retard senescence of plant tissues. Interestingly, the PLA2 products inhibited the activity of PLD, which has been suggested to be a key enzyme responsible for membrane lipid breakdown leading to plant senescence. Endogenous levels of lysophospholipids, such as lysophosphatidylethanolamine (LPE), could be increased in castor bean leaf discs by the treatment of auxin (50 μM), which is known to be a activator of PLA2. Pretreatment of leaf discs with a PLA2 inhibitor before auxin treatment nullified the auxin effect and rather resulted in accelerated senescence even compared to the nontreated control. Our recent results suggest a potential role of PLA2 products as biologically active molecules mediating hormonal regulation of growth and senescence. One such product LPE is being commercially exploited for retarding senescence and improving shelf life of fruits, vegetables, and cut flowers.

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Ethephon [2-(chloroethyl) phoshonic acid] is used widely to maximize the yield of ripe tomato fruit. However, ethephon causes rapid and extensive defoliation, overripening, and promotes sunscald damage to the fruit. Recent studies from our laboratory have provided evidence that lysophoshatidylethanolamine (LPE) can reduce leaf senescence. We investigated the potential use of LPE to reduce damaging effect of ethephon on tomato foliage. Three-month-old tomato plants (variety Mountain Spring) grown in greenhouse conditions were sprayed with 200 ppm LPE (with 3% ethanol) at 6 and 24 h before ethephon treatment. After 8 days, plants treated with ethephon alone showed about 80% foliar damage while plant treated with LPE before ethephon treatment showed about 25% foliar damage. In a parallel study, LPE together with ethephon was found to maintain three to four times greater chlorophyll content in the leaves compared to ethephon alone. Treatments of LPE did not reduce the fruit ripening response by ethephon. Both sources of LPE were effective in preventing damaging effects of ethephon on the foliage. These results suggest that LPE treatments 6 and 24 h before ethephon application can prevent damaging effects of ethephon on foliage while allowing the acceleration of fruit ripening.

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The pericarp tissue of red mature tomato (Lycopersicon esculentum cv. Gagliano) was used to exctract polygalacturonase (PG) enzyme. The technique for assaying PG activity involves measurement of released reducing groups that were linked together in pectin. Since the crude extract of PG from pericarp will contain considerable reducing groups, we found that repeated washings of the cell wall pulp removed much of the sugars and thus minimized the background absorbance without loss of PG activity. There is an inherent perplexity concerning the selection of blank for PG assay. This is because (i) the enzyme extract contains both the substrate (pectin) and product (free reducing groups) involved in the reaction; (ii) the color development with cyanoacetamide requires heating for 10 min. Thus, even though the reaction is terminated with borate buffer (pH 9.0) the breakdown of pectin continues chemically by heat; (iii) the absorbance from both pectin and enzyme together at zero time termination was always lower than the sum of absorbances from pectin alone and enzyme alone. This suggests that when together in the same tube, the enzyme appears to protect the pectin from physical breakdown during the period of 10 min. boil needed to develop color using the cyanoacetamide. Thus, the most appropriate blank is processing separately the solutions of enzyme alone and substrate pectin alone for color development and then adding the two absorbances. Using this improved assay we found that lysophosphatidylethanolamine (LPE) inhibited tomato PG activity. This inhibition appears to depend on the ripening stage of the fruit. Our results suggest that LPE is able to impart firmness to tomato fruit by reducing the PG activity, which in turn could protect the pectin/middle lamellae from enzymic breakdown. The effects of LPE on PG activity are distinct from those of Triton X-100 and lysophosphatidylcholine.

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