Project Abstract

<p>&nbsp; &nbsp;                <b>ABSTRACT</b></p><div>A postulated role of the CN-resistant alternative respiratory path-way in plants is the maintenance of mitochondrial electron trans-port at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) andsoybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19°C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28°C but was unchanged in soybean cotyledonsgrown at 14°C compared with plants grown at 28°C. When theshort-term response of tissue respiration was measured over thetemperature range of 35°C to 9°C, decreases in the activities of bothmain and alternative pathway respiration were observed regardlessof the growth temperature, and the relative partitioning of electronsto the alternative pathway generally decreased as the temperaturewas lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19°C supporting a role for the alter-native pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high an low temperature.</div><p></p>

Project Overview

<p> The biochemical basis of the CN-resistant alternative respiratory pathway in plants is an oxidase in the mitochondrial electron-transport chain that transfers electrons from reduced ubiquinone to oxygen, bypassing two sites of proton translocation and releasing the resulting free energy as heat (Moore and Siedow, 1991). Although the physio-logical role of this nonphosphorylating, energetically wasteful pathway remains unclear, a number of factors are known to affect alternative oxidase activity (Siedow and Umbach, 1995; Vanler berghe and McIntosh, 1997).&nbsp;</p><p>Regulation of alternative oxidase protein level through changes in gene expression and the dependence of activity on the reducing substrate, ubiquinol, have long been recognized(Moore and Siedow, 1991; Vanlerberghe and McIntosh,1997), but more recently two additional regulatory mechanisms have been identified.One regulatory mechanism is a redoxsensitive sulfhydryl/disulfide system, which, when oxidized, forms a disulfide bond between the subunits of the alternative oxidase homodimer, resulting in an essentially inactive enzyme (Umbach and Siedow, 1993).&nbsp;</p><p>The second regulatory feature involves activation by keto acids such aspyruvate (Millar et al., 1993), and the regulatory disulfide bond must be reduced for this latter activation to occur(Umbach et al., 1994). Recent studies using sulfhydryl re-agents have suggested that the site of keto acid action isat a sulfhydryl group, probably through formation of athiohemiacetal (Umbach and Siedow, 1996), and site-directed mutagenesis has been used to establish that both the regulatory sulfhydryl/disulfide system and the site of activation by keto acids involve the same Cysresidue(Rhoads et al., 1998). One of the consequences of these regulatory features is that the alternative oxidase can compete for electrons with an unsaturated Cyt pathway (RibasCarbo et al., 1995) rather than acting as a simple electron overflow path off the main Cyt pathway, as was postulated previously (Lambers, 1982).</p><p>Plant respiration rates are affected by numerous abiotic factors, and temperature is one of particular significance.There is a direct relationship between respiratory rate and temperature in the short term because the kinetics of most metabolic reactions are highly temperature dependent(Raison, 1980). In addition to short-term responses, plants grown at low temperatures often show higher rates of respiration than plants grown at higher temperatures when both are measured at the same temperature (Amthor, 1989;Collier and Cummins, 1990). This stimulation of respiration by growth at low temperatures has been reported to bean adaptive feature of plants grown in cold and arcticclimates compared with related species or ecotypes from warmer climates (Billings, 1974; McNulty and Cummins,1987). It has also been suggested that the increased rate of respiration at low temperatures involves a greater participation by the alternative pathway (McNulty et al., 1988;Purvis and Shewfelt, 1993).</p><p> </p><div>Plant growth at low temperatures often results in higherCN-resistant respiratory activity (McCaig and Hill, 1977;Elthon et al., 1986; McNulty and Cummins, 1987). This increased resistance to CN appears to be due to enhanced synthesis of alternative oxidase protein (Stewart et al.,1990; Vanlerberghe and McIntosh, 1992), as low tempera-ture increased the steady-state mRNA levels of aox1a and aox1b</div><div>&nbsp;genes in rice (Ito et al., 1997). Moreover, in isolated cells or in mitochondria, CN-resistant respiration is relatively insensitive to a wide range of temperatures (Yoshida and Tagawa, 1979; Stewart et al., 1990). Experiments with intact tissues have also concluded that the Cyt pathway is more sensitive to short-term changes in temperature than the alternative pathway (Collier and Cummins, 1990).</div><div><br></div><div>Chilling stress led to lower Cyt oxidase activity and protein levels in corn seedlings transferred to 14°C (Prasad et al.,1994) and in mung bean hypocotyls chilled at 0°C (Yoshidaet al., 1989). This suggests that at low temperatures the alternative pathway may be able to maintain a higher percentage of its relative activity than the Cyt pathway.Such alternative pathway activity may prevent the formation of potentially toxic active oxygen species that can result from over reduction of the ubiquin one pool following inhibition of the Cyt pathway (Purvis and Shewfelt, 1993;Wagner and Krab, 1995).</div><div><br></div><div> Previous attempts to measure the activity of the alternative pathway at low temperatures are suspect because the traditional use of inhibitors to assess the in vivo activities of the two electron-transfer pathways leads to in conclusive results if the pathways compete for electrons from the ubiquin one pool (RibasCarbo et al., 1995; Day et al., 1996).Furthermore, an increase in alternative oxidase protein levels will not necessarily lead to an increase in its activity in the absence of inhibitors. In tobacco leaves the level of the alternative oxidase protein was enhanced by adding salicylic acid, but neither the total respiratory activity nor the partitioning of electrons to the alternative pathway was affected by this treatment (Lennon et al., 1997).</div><div>In the present study we tested the hypothesis that low temperatures lead to greater alternative pathway activity in plants grown at either low (14°C or 19°C) or high (28°C)temperatures by measuring oxygen-isotope fractionation indifferent organs during tissue respiration over a temperature range from 9°C to 35°C. This technique allows in vivo measurements of the partitioning of electrons between the alternative and Cyt pathways in the absence of added inhibitors (Guy et al., 1989).<br></div><div><br></div><div> <div><b>MATERIALS AND METHODS</b></div><div><b><i>Plant Material</i></b></div><div>Mung bean (Vigna radiata[L.] Wilczeck) and soybean(Glycine max L. cv Ransom) seeds were treated with 0.5%NaHOCl for 10 min, washed, and hydrated in distilled water for 2 to 4 h with continuous air bubbling. Seeds were planted in a 1:1 mixture of gravel and sand and grown at a constant temperature of 19°C (mung bean), 14°C (soybean),or 28°C (both) in growth cabinets on a 14-h/10-h light/dark regime at 350 mol m2s1. The low-temperature treatments used in this study increased the time of germination and resulted in 2- to 3-fold slower growth relative to plants grown at the higher temperature for both mung bean and soybean. In addition, mung bean plants grown at temperatures below the 19°C used in this study were visibly damaged and did not survive beyond the first-leaf stage.</div><div><br></div><div>Based on the definition of stress as any external factor that exerts a disadvantageous influence, and on the fact that stress is most often measured in terms of factors that include growth (Taiz and Zeiger, 1998), both plant species were stressed when grown at the lower temperatures.Whether this specifically reflects changes in the balance between any components of the respiratory pathway is not known. Mung bean hypocotyls were harvested at d 15 in the 19°C temperature treatment at a developmental stage (first unfolding of primary leaves) that was equivalent to d 5 in the 28°C treatment. Sliced hypocotyl sections (0.8–1 cmlong) were used for respiratory measurements to minimize oxygen-diffusion limitations that may affect the isotope-fractionation measurements. Respiration of sliced hypocotyls was constant 10 to 15 min after the sections were made and remained so for several hours.</div><div>Leaf samples were taken from mung bean plants that had at least two fully expanded trifoliates at both growing temperatures. Three to four 10-cm2 discs of fully developed mung bean trifoliates were taken from the same plant for each experiment.Intact soybean cotyledons from plants grown at 14°C were collected at d 14 to 16, which was a develop mental stage (first unfolding of the primary leaves) equivalent to 6- to 7-d-old cotyledons of plants grown at 28°C</div> <br></div> <br> <br><p></p>