Measurement of the activity and capacity of the alternative pathway in intact plant tissues: Identification of problems and possible solutions

Three cytoplasmic male-sterile Nicotiana cultivars together with corresponding male-fertile progenitors and restored lines were investigated in order to find possible correlations between respiratory characteristics and male sterility. Oxygen consumption measurements were performed on cells from suspension cultures as well as on mitochondria isolated from green leaves. Inhibitors, which have been reported to specifically block either the cytochrome (KCN) or the alternative (propyl gallate and salicylhydroxamic acid [SHAM] respiratory pathways, were used in order to measure the capacity and activity of the two pathways. One of the inhibitors, SHAM, was found unsuitable to measure the activity of the altemative pathway due to the lack of specificity of SHAM for this pathway. A great difference in the capacity of the alternative pathway was detected between the two types of cell materials tested. Mitochondria isolated from green leaves showed a capacity of the alternative pathway of 5 to 20% of total mitochondrial repiration, while the capacity of cells from suspension cultures generally ranged from 50 to 80%. In addition to this, in organello synthesis of mitochondrial proteins revealed differences between mitochondria isolated from green leaves and from cell suspensions. No correlation, however, could be found between respiratory characteristics and male sterility.

Section: Discussionsupporting

confidence: 53%

Respiration in Cells and Mitochondria of Male-Fertile and Male-Fertile and Male-Sterile Nicotiana spp.

Håk̈ansson¹,

Glimelius²,

Bonnett³

1990

“…The results of a previous report indicated that estimates of the alternative pathway in intact tissues and in mitochondria both indicate higher levels in cold-grown shoots (7). Corn shoot tissues appear to satisfy the conditions set down by Moller et al (15) for using intact tissue measurements for estimating the alternative pathway. Thus, both intact corn tissues and mitochondria isolated from them can be used to measure alternative oxidase capacities.…”

mentioning

confidence: 56%

“…The role of this activity as a thermogenic process during reproductive development has been established as well as its induction by salicylic acid (16). During the current decade, attention has been directed to understanding the occurrence and function of the alternative oxidase in a wide variety of plant species and tissues, including crop species ( 15).…”

mentioning

confidence: 99%

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Stewart¹,

Martin²,

Reding³

et al. 1990

Respiration rates of Zea mays L. seedling tissues grown at 30 and 140C were measured at 250C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 300C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 140C, the increase in altemative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 140C after growth at 300C for 2 days had the same altemative oxidase capacity as seedlings grown at 300C. Seedlings grown at 140C for 10 to 12 days, then transferred to 300C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondna were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 200C. At 41°C, very little altemative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 410C was reversible. After incubation at 410C, the alternative oxidase capacity measured at 250C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 410C as they did when incubated at 250C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.

“…1C). Between 12 and 48 h, respiration rate grad- (18), but note that these terms are somewhat redefined here in that they may be measured in the presence of AA. All of the above data were calculated from the results presented in Figure 1.…”

Section: Respiratory Characteristics and Growthmentioning

confidence: 99%

Coordinate Regulation of Cytochrome and Alternative Pathway Respiration in Tobacco

Vanlerberghe

McIntosh²

1992

103

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA Mitochondrial electron transport in plants may proceed through either the CP2 or the AP (6,12,14). Transfer of electrons from NADH through the CP is coupled at three sites to the translocation of protons from the matrix to the intermembrane space. Reentry of protons to the matrix through complex V (mitochondrial ATP synthetase) results in the production of ATP. In this case, carbon oxidation in respiration is coupled to the production of ATP, and in many instances respiration rate is tightly regulated by the availability of ADP (i.e. adenylate control) (8, 12). Transfer of electrons through the AP bypasses two of the three sites of proton translocation. Under these conditions, carbon oxidation is not as tightly coupled to the production of ATP, and it is expected that respiration rate would not be as strictly regulated by the availability of ADP (8,12 It seems imperative that plants be able to regulate in a coordinate fashion the partitioning of electrons between the CP and the AP to meet changing metabolic requirements. Two important factors to be regulated will be, first, the capacity of each pathway to transfer electrons, and second, the degree to which that capacity is used (i.e. the degree of engagement of the pathway). The degree of engagement of the AP is dependent upon the redox poise of the ubiquinone pool (9) and, to some extent, upon the substrates being oxidized by the mitochondria (7).Although many studies indicate that plants are capable of altering the capacity of the AP (6, 14), little is known about the mechanisms by which this occurs. Some studies show that inhibition of the CP with respiratory inhibitors increases AP respiration. For example, treatment of rice roots with low concentrations of KCN or NaN3 for 6 h resulted in increased CN-resistant respiration, although it was not shown whether this CN-resistant respiration was sensitive to SHAM (11). In pea roots, a CN-resistant and SHAM-sensitive component of respiration was induced after several hours of KCN treatment (28). From the above reports, it is not clear whether the KCN treatment increased the capacity or simply the engagement of the AP. Also, interpretation of the data is hampered because KCN is known to inhibit many enzymes of metabolism (29). Nevertheless, inhibition of the CP by a specific respiratory inhibitor may be a useful tool to study mechanisms of induction of AP capacity in plants.AA is a specific inhibitor that blocks electron transfer between Cyt b and cl in the CP (20,23). In Euglena gracilis, addition of AA induced CN-resistant respiration within 5 h (2-4). A similar induction was seen in the yeast Hansenula anomala (17) and led to the ide...