Alternative Respiration

Root Research Introduction Virtual Course

Seminar

Summer 2002

Written by Elitsur Yaniv, ID 02525806-2

Tutor: Professor Amram Eshel

 

Contents

  1. Introduction
  2. Respiration - Mechanism
    1. Cytochrome Respiration Pathway
    2. Alternative Respiration Pathway
    3. Control
  1. Alternative Oxidation - Purposes
    1. pH Control
    2. Compensation for CN- Inhibition
    3. AOS Elimination
    4. Viability
    5. Thermogenesis
  1. Discussion
  2. Conclusions
  3. Bibliography

 

Introduction

Respiration is the breakdown of the six-carbon glucose molecule into smaller molecules while obtaining energy this way. The respiratory reactions sequence is common both to plants and animals. Respiration can be divided into two main steps. The first one is the glycolysis while the following one is Krebs cycle. Glyclysis takes place within the cytosol while Krebs cycle takes place within the mitochondria. Glycolysis is typical to all living creatures while Krebs cycle is only typical to aerobic creatures. Either Oxygen consumption or Carbon Dioxide production can be used in order to measure the rate of this sequence, since both occur during respiration.      

Both steps mentioned in the previous paragraph are subdivided into further steps. Needless to mention the necessity of enzymes in order to catalyze all these reactions.

The terminal part of respiration is the Electron Transport Chain. During Krebs cycle accumulation of NADH takes place within the mitochondrion. Later with the assistance of oxygen, the Electron Transport Chain takes place at the inner membrane of the mitochondrion.

Respiration can be classified to two pathways. The pathway common to all living organisms is called cytochrome respiration while the pathway occurring in plants, many algae, fungi and some protozoa is called alternative respiration.

Although many articles claim that no certain function of the alternative respiration pathway was determined, many functions are related to it. Alternative respiration occurs in most plant organs. Some of its functions are not organ specific while others are. Alternative respiration is especially active in roots due to its relation to oxygen uptake, which is the result of phosphate uptake occurring in roots.

Respiration - Mechanism

Both the cytochrome respiration pathway and the alternative respiration pathway begin at Protein Complex I, when NADH is being oxidized. One proton is transported by Complex I to the inter membrane space, while two electrons are transported within the inner membrane by the ubiquinone. The ubiquinone loaded with these electrons, or in other words at its reduced state (Qr), transfers these electrons either to complex III or to the Alternative Oxidase (AOX).

Due to the fact that the ubiquinone is the point in which the reaction can proceed in different ways, it is called the branch point.

Cytochrome Respiration:

 

Fig 1 - Cytochrome respiration pathway.

 

The cytochrome respiration pathway is typical to all living organisms. It proceeds while Complex III pulls out another proton from the matrix to the inter membrane space. The electrons are taken by cytochrome C who crawls upon the outer side of the inner membrane towards Protein Complex IV. Complex IV pulls out another proton similarly to Complexes I and III and transports the electrons back into the inner domain of the mitochondrion. At this stage oxygen is consumed with a proton and these two electrons to yield water.

When the electron arrives at the inner domain of the mitochondrion it reacts with oxygen and a couple of protons. The product of this reaction is H2O.

During the Electron Transport Chain up to the branch point and at the standards respiration pathway from the branch point onwards, a proton gradient (Meaning H+ ions gradient) is maintained having many more protons within the inter membrane space in relation to the matrix. This gradient facilitates ATP production performed by the ATP Synthase Complex.

The cytochrome respiration pathway can be inhibited with azide, CO, but the most famous inhibitor, which activates our imagination on issues such as spying and war stories, is the cyanide.

Alternative Respiration:

Fig 2 - Alternative respiration pathway.

 

According to Biochemistry & Molecular Biology of Plants, alternative respiration is typical to plants, many algae, fungi and some protozoa. It proceeds from the branch point in a different manner than the cytochrome respiration pathway. The alternative pathway does not contribute to the proton gradient. It consumes oxygen and oxidizes NADH to NAD+, but it does not contribute to the proton gradient and therefore, it does not lead to the production of ATP molecules.

 

Fig 3 - Alternative oxidase - inactive and active forms.

The protein performing the alternative respiration is the Alternative Oxidase (AOX). The AOX is a dimer at its inactive (oxidized) form. The dimer is split to two proteins at its active (reduced) form.

For the purpose of transporting the electrons, the AOX has a putative iron binding site at its C terminal and the cysteine peptide which enables the formation of the active and inactive forms is located towards the N terminal.

Fig 4 - Alternative oxidase - structure : emphasis on the iron binding site and the cysteine dimerization peptide.

During the alternative respiration process oxygen is consumed and through a reaction with the electrons transported to the AOX and a proton, water is yielded.

If we compare the alternative respiration pathway to the cytochrome respiration pathway, we can grasp the alternative pathway as a bypass. Both pathways transfer a proton or protons to the inter membrane space, they transfer a couple of electrons and consume oxygen to yield water. The difference is that the cytochrome pathway transports two additional protons from the matrix to the inter membrane space, and therefore it enables a greater proton gradient for the same effort. The fact that eventually less energy is obtained trough the alternative respiration pathway makes us wonder why is this pathway necessary.

According to Biochemistry & Molecular Biology of Plants, Alternative respiration isn't inhibited by cyanide and the rest of the cytochrome pathway inhibitors. Therefore their inhibition is performed after the branch point, namely at the cytochrome C. The alternative pathway inhibitors are: saliecylhydroxamic acid (SHAM) and n-propylgallate.

Fig 5 - Alternative oxidase inhibitors - saliecylhydroxamic acid (SHAM) and n-propylgallate.

 

Control:

Fig 6 - Alternative oxidation regulation models.

 

Two models are presented in Biochemistry & Molecular Biology of Plants, according to which the decision is made whether to take the cytochrome respiration pathway or the alternative respiration pathway.

 

The first model (A) claims that the alternative respiration pathway is taken only when the cytochrome respiration pathway is saturated. The decision is taken according to the Qr/Qt ratio, meaning the rate of reduced ubiquinone, not free to transport electrons in relation to the total amount of ubiquinones - reduced or oxidized. When the Qr/Qt ratio is high, meaning that not many ubiquinones are free to transport electrons, then the alternative respiration pathway would proceed.

The second model (B) claims that alternative oxidation proceeds at a lower Qr/Qt ratio. Alternative oxidation is controlled by α-keto acids and by the redox state of the AOX disulfide bonds. This model assumes the existence of another mechanism not yet determined.

Today the second model is believed to be the correct one. Additionally, it is believed that cytochrome respiration is blocked in situation of proton excess at the inter membrane space in relation to the matrix.

Alternative Oxidation - Purposes

pH Control:

One generalist assumption, discussed in Plant and Cell Physiology, not specific to roots is pH control, since complex I transports protons to the inter membrane space, and thereby pH depends also on complex I activity. Since AOX encourages additional activity of complex I, pH control assumption was assumed.

In a state of proton surplus alternative respiration can facilitate in eliminating these unwanted protons less limited by factors such as proton gradient than the cytochrome respiration.

This is an interesting assumption, which indeed needs to be further analyzed.

Compensation for CN- Inhibition:

Another generalist assumption is that since the AOX is cytochrome respiration inhibitors resistant, it can partially compensate for damages done by such. It does contribute to the proton gradient, and in turn, to ATP synthesis.

The issue is discussed briefly in the Journal of Experimental Botany, where less sensitivity towards cyanide in mentioned as the result of increase in the AOX activity due to citrate.

This assumption is very limited, since it can only be right for very minute amounts of CN-.

AOS Elimination:

An issue directly related to roots (Together with viability) is AOS elimination, which is related to phosphate uptake. It is reviewed in Physiologia Plantarum (October 2001 issue) in an extensive and specific article as well as in the Proceeding of the National Academy of the United States in a more general article that does not focus on roots.

AOS are the initials of Active Oxygen Species. They exhibit toxicity characteristics, and therefore the organism has to develop mechanisms to eliminate them. Statistically, AOS are present amongst the non-AOS oxygen molecules. When too much oxygen is present probability is higher that there will be AOS. AOS molecules can be eliminated by chemical reaction with two protons and two electrons to yield H2O.

The reason for which oxygen penetrates roots is phosphate uptake. Phosphate is one of the most important mineral nutrients for living organisms and it can be the organism's growth-limiting factor.

Fig 7 - Respiration (O2) uptake and phosphate uptake (Upper line) per time.

 

In an experiment on bean plants' roots, it has been observed that oxygen uptake is tightly correlated to phosphate uptake in an experiment conducted on tobacco plants (Fig 7).

Phosphate uptake is increased when there is a phosphate stress condition. The plant needs to put more effort in order to get the amount of phosphate it needs. And then as previously said oxygen uptake is increased for which AOS are more probably to be present.

 

Viability:

In addition to AOS elimination, viability - discussed in Physiologia Plantarum (July 2001 issue) - is another AOX issue related to roots.

In botany, the best way to find out about survival advantages having a certain component is by suppressing it. Suppression of AOX is performed by AS8, which is the AOX anti-sense.

It can be seen on table 1 that the wt viability rates are higher in relation to the mutant and that the mutant's dry weight is higher in relation to the wt.

The assumption is that the AOX increases the survival rates, and that on phosphate stress conditions the survival strategy would be to limit the growth rate.

Table 1 - Viability and dry weight per time on a P- culture.

 

Thermogenesis:

Biochemistry & Molecular Biology of Plants discussed a very interesting function related to alternative respiration. Although not related to roots I’ve chosen to mention thermogenesis (Meaning increase in temperature) preformed by the AOX. In this respect it would be appropriate to distinguish thermogenic floral organ plants from plants lacking thermogenic floral organs. Water lilies are considered to be thermogenic organ flower plants. These thermogenic organs can raise their temperature by 10°C above the temperature of its surrounding. As for non-thermogenic organ flower plants, their temperature can be raised by only hundredths of degrees as a result of the AOX reaction.

 

Fig 8 - Thermogenic appendix

 

Not all the energy available is used to increase the proton gradient, and excess energy is released as heat. The reason that thermogenic plants raise their floral organs temperature in rates much higher in comparison to non-thermogenic plants is due to the presence of many more mitochondria within the thermogenic organs. Thermogenic plants such as voodoo lilies produce a club-like structure (Called an appendix) which is the thermogenic part of the flower.

The purpose of thermogenesis in plants is volatilizing chemicals and thereby attracting pollinators or it can be carrion mimicry on which eggs are planted by certain insects in order to attract these insects.

Discussion

According to articles concerning the alternative respiration pathway not all the AOX purposes were determined yet, which makes it a very interesting issue.

The AOX is related to survival strategies whether it is concerning roots or other organs such as flowers.

AOS elimination during increased phosphate uptake, in response to phosphate stress condition is a key survival issue. Since phosphate is one of the growth-limiting factors, increased uptake is a necessity in such conditions, and the byproduct, namely AOS should be eliminated in this case.

Thermogenesis, related to floral organs, can be seen as a luxury, yet many survival techniques might have been seen as unnecessary characteristics at their beginning and eventually brought to conquest of additional habitats.

Issues which are not organ related nor fully understood such as pH control and compensation for CN- inhibition are needed to be further analyzed and differences in relation to organs may by found when these functions are better understood.

Conclusions

The best conclusion I can draw from writing this seminar is that organ related characteristics are found in two ways. It can be found through the fact that there are different characteristics in relation to different organs. The other option is by recognizing a characteristic typical to a certain cell component and when deeply understanding it, it can be attributed to certain organs in relation to other organs. Obviously, the first method is top-down, meaning from the phenotype level to the cellular level. The second method is bottom-up, from the cellular level to the phenotypic level.

Bibliography

  1. Buchanan, Gruissem and Jones, Biochemistry & Molecular Biology of Plants 696-705
  2. Denis P. Maxwell, Yong Wang, and Lee McIntosh, Proceeding of the National Academy of the United States 96: 8271-8276, July 1999 [Full Text] [PDF]
  3. Frank Millenaar, Utrecht University [PDF]
  4. Frank F. Millenaar, Miquel A. Gonzalez-Meler, James N. Siedow, Anneke M. Wagner, and Hans Lambers, Journal of Experimental Botany 53(371): 1081-1088, May 2002 [Full Text] [PDF]
  5. Hannah L. Parsons, Justine Y.H. Yip, and Greg C. Vanlerberghe, Plant Physiology 121: 1309-1320, December 1999 [Full Text]
  6. Izabela M. Juszczuk, Anneke M. Wagner, Anna M. Rychter, Physiologia Plantarum 112(3): 185-192, October 2001 [Full Text] [PDF]

7.      Justine Y. H. Yip and Greg C. Vanlerberghe: Physiologia Plantarum, 112 (3): 327, July 2001[Abstract] [Full Text]

  1. Sakano K, Plant and Cell Physiology, 39 (5): 467-473, May 1998 [Abstract]