Alternative Respiratory Pathways in Higher Plants E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 787

Veröffentlichungsjahr: 2015

Beschreibung

Rapid developments in molecular and systems biology techniques have allowed researchers to unravel many new mechanisms through which plant cells switch over to alternative respiratory pathways.

This book is a unique compendium of how and why higher plants evolved alternative respiratory metabolism. It offers a comprehensive review of current research in the biochemistry, physiology, classification and regulation of plant alternative respiratory pathways, from alternative oxidase diversity to functional marker development. The resource provides a broad range of perspectives on the applications of plant respiratory physiology, and suggests brand new areas of research.

Other key features:

• 
  written by an international team of reputed plant physiologists, known for their pioneering contributions to the knowledge of regular and alternative respiratory metabolism in higher plants

• 
  includes step-by-step protocols for key molecular and imaging techniques

• 
  advises on regulatory options for managing crop yields, food quality and environment for crop improvement and enhanced food security

• 
  covers special pathways which are of key relevance in agriculture, particularly in plant post-harvest commodities

Primarily for plant physiologists and plant biologists, this authoritative compendium will also be of great value to
postdoctoral researchers working on plant respiration, as well as to graduate and postgraduate students and university staff in Plant Science. It is a useful resource for corporate and private firms involved in developing functional markers for breeding programs and controlling respiration for the prevention of post-harvest losses in fruit, vegetables, cut flowers and tubers.

Leseprobe

Alternative respiratory pathways in higher plants

EDITED BY

Kapuganti Jagadis Gupta

Department of Plant SciencesUniversity of OxfordOxford, UK

Luis A.J. Mur

Institute of BiologicalEnvironmental and Rural ScienceAberystwyth UniversityAberystwyth, UK

Bhagyalakshmi Neelwarne

Plant Cell and Biotechnology DepartmentCSIR-Central Food Technological Research InstituteMysore, India

This edition first published 2015 © 2015 by John Wiley & Sons, Ltd

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Library of Congress Cataloging-in-Publication Data:

Gupta, Kapuganti Jagadis   Alternative respiratory pathways in higher plants / Kapuganti Jagadis Gupta, Luis A.J. Mur, and Bhagyalakshmi Neelwarne.         pages   cm   Includes bibliographical references and index.

   ISBN 978-1-118-79046-5 (cloth)1. Plants–Respiration.   2. Plant genetics.   3. Plant physiology.   I. Mur, Luis A. J.   II. Neelwarne, Bhagyalakshmi.   III. Title.   IV. Title: Respiratory pathways in higher plants.   QK891.K37 2015   581.3′5–dc23

               2014050165

A catalogue record for this book is available from the British Library.

Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books.

Cover image: Main cover picture created by Birgit Arnholdt Schmidt and Kapuganti Jagadis Gupta

List of contributors

Salvador Abreu

Department of Computer Science, Universidade de Évora, Évora, Portugal

Mary S. Albury

Biochemistry and Molecular Biology, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK

Birgit Arnholdt-Schmitt

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Natalia V. Bykova

Cereal Research Centre, Agriculture and Agri-Food Canada, Morden, MB, Canada

Maria Doroteia Campos

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Hélia G. Cardoso

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

José Hélio Costa

Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Ceara, Brazil

Marina Cvetkovska

Department of Biological Sciences and Department of Cell and Systems Biology, University of Toronto Scarborough, Toronto, Ontario, Canada

Keshav Dahal

Department of Biological Sciences and Department of Cell and Systems Biology, University of Toronto Scarborough, Toronto, Ontario, Canada

Matthias Döring

INOQ GmbH, Solkau, Schnega, Germany

Padmanabh Dwivedi

Department of Plant Physiology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India

Catherine Elliott

Biochemistry and Molecular Biology, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK

Margarida Espada

NemaLab-ICAAM, Departamento de Biologia, Universidade de Évora, Évora, Portugal

Ralph Ewald

Institut für Biowissenschaften, Abteilung Pflanzengenetik, Universität Rostock, Rostock, Germany

António Miguel Frederico

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Teresa Gonçalves

Department of Computer Science, University of Évora, Évora, Portugal

Kapuganti Jagadis Gupta

Department of Plant Sciences, University of Oxford, Oxford, UKCurrent address: National Institute of Plant Genome Research, Aruna Asaf Ali Road, New Delhi, India

Lee D. Hansen

Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah, USA

Renate Horn

Institut für Biowissenschaften, Abteilung Pflanzengenetik, Universität Rostock, Rostock, Germany

Abir U. Igamberdiev

Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada

Jens Jurgeleit

INOQ GmbH, Solkau, Schnega, Germany

Sarma Rajeev Kumar

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, India

Wu-Sheng Liang

Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, People’s Republic of China

Eva Lucic

INOQ GmbH, Solkau, Schnega, Germany

Allison E. McDonald

Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada

Kaveh Mashayekhi

BioTalentum Ltd, Budapest, Hungary

Ben May

Biochemistry and Molecular Biology, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK

Dirce Fernandes de Melo

Department of Biochemistry and Molecular Biology, Federal University of Ceara, Fortaleza, Ceara, Brazil

Louis Mercy

INOQ GmbH, Solkau, Schnega, Germany

Anthony L. Moore

Biochemistry and Molecular Biology, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK

Manuel Mota

NemaLab-ICAAM, Departamento de Biologia, Universidade de Évora, Évora, Portugal

Luz Muñoz-Sanhueza

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal Current address: Department of Plant and Environmental Sciences (IPM), Norwegian University of Life Sciences, Ås, Norway

Luis A.J. Mur

Institute of Biological, Environmental and Rural Science, Aberystwyth University, Aberystwyth, UK

Bhagyalakshmi Neelwarne

Plant Cell and Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore, India

Tânia Nobre

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Carlos Noceda

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal Current address: Prometeo Project (SENESCYT), CIBE (ESPOL), Guayaquil, Ecuador

Amaia Nogales

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Halley Caixeta Oliveira

Departamento de Biologia Animal e Vegetal, Centro de Ciências Biológicas, Universidade Estadual de Londrina (UEL), Londrina, Paraná, Brazil

Michail Orfanoudakis

Department of Forestry and Management of the Environment and Natural Resources, Forest Soil Lab, Democritus University of Thrace, Orestiada, Greece

Augusto Peixe

Melhoramento e Biotecnologia Vegetal, ICAAM, Universidade de Évora, Évora, Portugal

Alexios Polidoros

Department of Genetics and Plant Breeding, School of Agriculture, Aristotle University of Thessaloniki, Thessaloniki, Greece

Paulo Quaresma

Department of Computer Science, University of Évora, Évora, Portugal

Carla Ragonezi

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Anna M. Rychter

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland

Ione Salgado

Departamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), São Paulo, Brazil

Elisete Santos Macedo

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Ramalingam Sathishkumar

Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, India

Caroline Schneider

INOQ GmbH, Solkau, Schnega, Germany

Samir Sharma

Department of Biochemistry, University of Lucknow, Lucknow, India

Evangelia Sinapidou

Department of Agricultural Development, Democritus University of Thrace, Orestiada, Greece

Debabrata Sircar

Biotechnology Department, Indian Institute of Technology Roorkee, Uttarakhand, India

Jan T. Svensson

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal Current address: Nordic Genetic Resource Center, Alnarp, Sweden

Bożena Szal

Institute of Experimental Plant Biology and Biotechnology, Faculty of Biology, University of Warsaw, Warsaw, Poland

Vera Valadas

EU Marie Curie Chair, ICAAM, Universidade de Évora, Évora, Portugal

Greg C. Vanlerberghe

Department of Biological Sciences and Department of Cell and Systems Biology, University of Toronto Scarborough, Toronto, Ontario, Canada

Cláudia Vicente

NemaLab-ICAAM, Departamento de Biologia, Universidade de Évora, Évora, Portugal

Jia Wang

Department of Biological Sciences and Department of Cell and Systems Biology, University of Toronto Scarborough, Toronto, Ontario, Canada

Luke Young

Biochemistry and Molecular Biology, School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK

Preface

Respiration is a crucial biochemical process found in all living organisms for meeting their energy demands. A cell adapts to its surroundings and dynamically caters to the energy needs of a wide array of functions. Thus, cells have evolved mechanisms to ingeniously ‘switch on’ and ‘switch off’ the different steps of respiratory mechanisms. Among the biochemical processes involved in respiration, three major highly conserved ‘classical’ pathways are involved; glycolysis, where energy is generated by breaking down glucose; the tricarboxylic acid (TCA) cycle, where the energy is generated in a form that can be used in cellular biochemical reactions; and electron transfer through an electron transport chain to form reducing equivalents leading to the generation ATP. Additionally, plant cells can regulate respiration in a manner deviating from fundamental and generic pathways via so-called alternative respiratory pathways (ARP), which form the focus of this book. While alternative modes of respiration occur in parallel to normal respiration, different sets of regulatory mechanisms are involved in the regulation of genes encoding for the proteins that are involved in alternative pathways. Understanding the regulation of these genes is an important theme in ARP research. Thus, the means through which alternative respiratory processes are regulated to help maintain classical respiration under various stresses or during discrete developmental or ecological conditions, features prominently in ARP publications. Linked to such research are attempts to predict the responses to climate change – changes in temperature, gases, physical vibrations, light, cosmic energy and so on. Even at the shortest and smallest scales, the plant’s immediate environment directly influences in planta physiological processes – via processes such as respiration – which are ultimately regulated at the genetic level. As a result, on longer and larger spatiotemporal scales, such environmental effects bring about changes in the distribution of plant species and ecosystems. Such changes will in turn also impact on the climate through the exchange of energy and gases among the flora and fauna around them. Equally, a failure to understand and respond to the impacts of climate change on respiration in crops will compromise yield, perturbing food security. Aware of these facts, plant physiologists have focused their research into each aspect of these interactions. A great deal of research has recently been published on how plants display different modes of respiration in different organs by switching over to ARP and on what set of parameters regulate alternative oxidases. To highlight the contribution of ARP to these fundamentally important topics we have brought together scientists with global reputations in the field to produce what we consider to be an important book with relevance to ecology, plant biodiversity and crop production.

This book therefore considers both classical and alternative respiratory pathways in diverse plant species and in different organs of the same plant at different times of its life cycle. Another driving principle has been to consider the potential applications of this knowledge to plant science and agriculture. The sixteen chapters are split into three sections: the first shows how plant respiratory mechanism have developed to thrive by cleverly rationing cellular energy under differing circumstances, while the second section highlights the application of ARP in plant breeding. The book wraps up the third and final section with the description of important protocols that will be useful for newer researchers.

Within Section A, Chapter 1 introduces readers to the basic principles and the principal difference between classic respiration and the alternative respiratory mechanisms. Complex regulatory mechanisms are described indicating the possibility of not only switching from glycolysis to fermentative metabolism but also the utilization of ARP to maintain substrate oxidation while minimizing the production of ATP. Equally, new insights are indicated on how ATP generation can be maintained under hypoxia. Chapter 2 describes the uncoupling pathways of plant mitochondrial electron transport and the mechanisms variously evolved to maintain the energy flux. How the regulatory proteins – the alternative oxidases – are distributed among the plant kingdom is brought into focus in Chapter 3.

Chapters 4 to 9 deal with alternative respiration under endogenous biochemical perturbations that occur due to certain signal molecules and exogenous stress, as well as how mitochondrial metabolism is regulated and cellular energy is balanced. Chapters 10 and 11 specifically address certain issues related to horticultural commodities – ARP in fruit ripening and in bulky storage tissues.

Section B contains subsections 12 to 14 – a package of 12 chapters – that consider how the molecular information on alternative oxidases may be developed as functional markers in plant breeding programmes. In-depth information is provided by the most renowned experts in the field, discussing how alternative oxidase genes also serve to develop phenotyping tools based on calorespirometry. Since alternative respiratory pathways play a role in the generation of heat during flower blooming and fruit ripening – where heat is needed for emitting volatiles – it serves as an excellent tool for calorespirometric measurements of metabolic heat rates and carbon dioxide rates of respiring tissues as functions of temperature. This enables the rapid responses of plant metabolic events to temperature fluctuations to be determined and, therefore, plant adaptability to environmental conditions to be deduced. Investigating such responses often involves cumbersome and expensive experiments which may be avoided by opting for methods such as calorespirometry. This area has great potential for projecting the effects of global warming on the plant kingdom as a whole and for predicting the geographical distribution of different crops and plant species.

Section C, which includes Chapters 15 and 16, provides updated protocols that describe the steps involved in the isolation of mitochondria for different studies, written by the most experienced workers in the field.

This book, with its breadth of information from the classical understanding of plant respiratory mechanisms to the highly specialized physiological changes that occur in plants during ARP, is expected to find a large readership among life science students and researchers in plant science.

Reputed scientists from nine different countries have contributed to this book and to whom we editors are extremely grateful. We owe our heartfelt gratitude to the internal editors and book publishing staff of John Wiley & Sons, Ltd. for their continuous support and timely advice during the course of the preparation of this volume.

K.J. Gupta, L.A.J. Mur and B. Neelwarne

SECTION APhysiology of plant respiration and involvement of alternative oxidase

Contents

1 Integrating classical and alternative respiratory pathways

Kapuganti Jagadis Gupta, Bhagyalakshmi Neelwarne and Luis A.J. Mur

2 Non-coupled pathways of plant mitochondrial electron transport and the maintenance of photorespiratory flux

Abir U. Igamberdiev and Natalia V. Bykova

3 Taxonomic distribution of alternative oxidase in plants

Allison E. McDonald

4 Alternative pathways and phosphate and nitrogen nutrition

Anna M. Rychter and Bożena Szal

5 Structural elucidation of the alternative oxidase reveals insights into the catalytic cycle and regulation of activity

Catherine Elliott, Mary S. Albury, Luke Young, Ben May and Anthony L. Moore

6 The role of alternative respiratory proteins in nitric oxide metabolism by plant mitochondria

Ione Salgado and Halley Caixeta Oliveira

7 Control of mitochondrial metabolism through functional and spatial integration of mitochondria

Samir Sharma

8 Modes of electron transport chain function during stress: Does alternative oxidase respiration aid in balancing cellular energy metabolism during drought stress and recovery?

Greg C. Vanlerberghe, Jia Wang, Marina Cvetkovska and Keshav Dahal

9 Regulation of cytochrome and alternative pathways under light and osmotic stress

Padmanabh Dwivedi

10 Alternative respiratory pathway in ripening fruits

Bhagyalakshmi Neelwarne

11 Respiratory pathways in bulky tissues and storage organs

Wu-Sheng Liang

CHAPTER 1Integrating classical and alternative respiratory pathways

Kapuganti Jagadis Gupta1,*, Bhagyalakshmi Neelwarne2 and Luis A.J. Mur3

1Department of Plant Sciences, University of Oxford, Oxford, UK

2Plant Cell and Biotechnology Department, CSIR-Central Food Technological Research Institute, Mysore, India

3Institute of Biological, Environmental and Rural Science, Aberystwyth University, Aberystwyth, UK

*Current address: National Institute of Plant Genome Research, Aruna Asaf Ali Road, New Delhi, India

Introduction

Respiratory pathways are vital for plant carbon and energy metabolism, which is the main use of most assimilated carbohydrates. Most respiratory pathways are very well established, the prominent being glycolysis in cytosol and the tricarboxylic acid (TCA) cycle, which occurs in the matrix of mitochondria coupled with the electron transport chain (ETC) which functions along the inner mitochondrial membrane. Some glycolytic enzymes also associate with the mitochondrial membrane and dynamically support substrate channelling (Giegé et al., 2003; Graham et al., 2007). Despite cross-kingdom commonalities in glycolysis and the TCA cycle, the regulation of respiration is relatively poorly understood (Fernie et al., 2004) which reflects the complexity of respiratory pathways. In plants this complexity encompasses the only possibility of switching from glycolysis to fermentative metabolism but the utilization of alternative pathways in plants allows the maintenance of substrate oxidation while minimizing the production of ATP. Equally, new insights have suggested how ATP generation can be maintained under hypoxia. With this overview, this chapter will integrate such alternative respiratory pathways with components of the classical oxidative-phosphorylative pathways.

Mitochondrial electron transport generates ATP by using the reducing equivalents derived through the operation of the TCA-cycle. The classic operation of the ETC pathway involves the transport of electrons from such as NAD(P)H or succinate to oxygen via four integral membrane oxidoreductase complexes: NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), cytochrome c reductase (complex III), cytochrome c oxidase (complex IV or COX), linked to a mobile electron transfer protein (cytochrome c) and ATP synthase complex (complex V). In complex V, the active extrusion of protons from the inner membrane space to the matrix leads to the generation of ATP (Boekema and Braun, 2007) (Figure 1.1). Apart from this classical operation of the ETC, mitochondrial complexes interact to form so-called super-complexes or respirosomes (Boekema and Braun, 2007). Complex I, II and IV are involved in the formation of super-complexes with different degrees and configurations. It may be that the formation of super-complexes represents a regulatory mechanism that controls the passage of electrons through the ETC (Eubel et al., 2003). Super-complex formation helps in increasing the stability of individual complexes, in the dense packing of complexes in the membrane and in fine tuning energy metabolism and ATP synthesis (Ramírez-Aguilar et al., 2011).

Figure 1.1 Overview of electron transport chain dissipatory mechanisms in plant mitochondria.

Currently most research on alternative electron transfer is focused on non-phosphorylating bypass mechanisms: a second oxidase – the alternative oxidase (AOX), an external NAD(P)H dehydrogenases in the first part of ETC, and also plant uncoupling mitochondrial proteins (PUCPs).

Alternative oxidase (AOX)

AOX is located in the inner mitochondrial membrane of all plants and fungi and a limited number of protists. AOX also appears to be present in several prokaryotes and even some animal systems (Chaudhuri and Hill, 1996; McDonald, 2008; McDonald and Vanlerberghe, 2006). Two forms of AOX are present in dicot plants (AOX1 and AOX2) while in monocots there is only one AOX (AOX1) (Considine et al., 2002; Karpova et al., 2002).

AOX are homodimeric proteins orientated towards the inner mitochondrial matrix. AOX diverts electrons from the main respiratory chain at the ubiquinone pool and mediates the four-electron reduction of oxygen to water (Figure 1.1). In comparison to electron transfer by the cytochrome chain (complex III and IV), AOX does not pump H+, therefore transfer of electrons by AOX does not create a transmembrane potential, and the decline in free energy between ubiquinol and oxygen is dissipated and mostly released as heat (Vanlerberghe et al., 1999). The diversion of electrons to the AOX pathway can reduce ATP generation by up to 60% (Rasmussen et al., 2008). The AOX ATP dissipatory pathway plays an important role when the ETC is inhibited by various stress conditions. ETC inhibition increases NADH/NAD+ and ATP/ADP ratios and as a consequence the TCA cycle could slow down. In addition to the energetic consequences of this, the number of carbon skeletons being produced will also be limited as the export of citrate supports nitrogen assimilation. Against this, AOX contributes to the maintenance of electron flow and the production of reducing equivalents to help maintain the TCA cycle. Indeed, AOX activation occurs in direct response to stress. A feature of all stress conditions is an increase in the production of reactive oxygen species (ROS): a process that can occur from the over-reduction of cytochrome components through the disruption of the ETC. In response to this, ROS or ROS-induced signals such as salicylic acid, act to induce the transcription of AOX (Vanlerberghe and McIntosh, 1997; Mackenzie and McIntosh, 1999) as also suggested from the observation that the addition of antioxidants leads to the suppression of AOX (Maxwell et al., 2002).

Oxygen, AOX and COX

Once induced by ROS, AOX may function as a negative feedback mechanism to suppress ROS production; a feature that we have named oxygen homeostasis (Gupta et al., 2009). This feedback mechanism is a consequence of large differences in O2 affinities of the classical and alternative respiratory pathways. The Km of COX is approximately 0.1 μmol but in AOX it is between 10 and 20 μmol (although the study by Millar et al., 1993 suggested a 10-fold higher AOX affinity for O2). Given these affinities, COX will maintain respiration whilst AOX reduces the O2 concentration, thereby decreasing the production of ROS inside the mitochondrion (Puntarulo and Cederbaum, 1988; Skutnik and Rychter, 2009). This is supported by the observations of Ribas-Carbo et al. (1995) who used an oxygen isotope discrimination technique to show that the inhibition of AOX by its inhibitor salicylhydroxamic acid (SHAM) did not lead to a decrease in total respiratory rates. This mechanism would be an exception to the ‘energy over flow’ model proposed by Lambers (1982), who suggested that in certain situations (e.g. excess carbohydrate), non-phosphorylating alternative pathways might contribute significantly to total respiration. Oxygen homeostasis could be of especial relevance in situations where different plant tissues are subjected to fluctuating O2 concentration due to diffusion gradients, and more so under environmental conditions such as flooding (Rolletschek et al., 2002; Bailey-Serres and Chang, 2005; Schmälzlin et al., 2005; Bailey-Serres and Voesenek, 2008; Rasmusson et al., 2008).

The electron partitioning model of Ribas-Carbo et al. (1995) suggests that COX and AOX compete for electron and electron passage but this must be influenced by the stress response of each pathway and particularly if exposed to low partial pressures of O2 (Po2). In a study undertaken by the senior author’s group, root slices of several species were incubated in a sealed cuvette and the respiratory rate of the tissue was measured until total oxygen was depleted in the vial. Until a partial pressure of 4% Po2, the decrease in respiratory rate correlated linearly with O2 concentration; however, at <4% Po2 level, the respiratory oxygen consumption rate slowed, taking a longer time to consume oxygen, indicating that a more slowly respiring plant would promote survival under the latter condition (Zabalza et al., 2009). This unique phenomenon has been named as the ‘adaptive response of plant respiration (ARPR) to hypoxia’. To determine which among the respiratory pathways could be influencing ARPR, each pathway was selectively inhibited in hydroponically grown pea using either KCN (an inhibitor for COX) or SHAM (an inhibitor for AOX). When AOX was the only electron acceptor, O2 consumption continued without any alteration until all the oxygen was depleted, but when AOX was inhibited, ARPR was still observed. Thus, the COX pathway was found to be responsible for ARPR (Zabalza et al., 2009). Clearly, ARPR is not a consequence of differentially responsive O2 affinities of the terminal oxidases (see earlier) as it occurs at Po2 above the Km of both oxidases. The decline in respiration could not be explained by a depletion of carbohydrates, as respiratory substrates, since when the same root material was immediately reused in experiments, ARPR was still observed. Moreover, oxygen diffusion through the tissue was not limiting at low Po2 because ARPR was also observed with in single-celled organism Chlamydomonas which has a diameter approximately 20 μm (Gupta et al., 2009). The lower Po2 was not in itself limiting respiratory rates as respiration could be elevated by the prior addition of 10 mM pyruvate prior to assessing ARPR. Taken together, these observations point towards the most likely scenario of the existence of an oxygen sensing mechanism that regulates the rate of mitochondrial oxygen consumption at low Po2.

Pyruvate kinases, classical respiratory metabolism and AOX

Pyruvate kinase (PK; EC 2.7.1.40) plays a critical role in glycolytic pathway catalyzing the terminal reaction of the glycolytic pathway by converting ADP and phosphoenolpyruvate (PEP) to ATP and pyruvate. As pyruvate regulates both glycolysis and the TCA cycle (Pilkis and Granner, 1992; Teusink et al., 2000), PK represents a crucial respiratory regulatory node. PK exists as tissue-specific isozymes that exhibit significant differences in their physical and kinetic properties (reviewed by Plaxton and Podesta, 2006). This reflects the presence of different PK isozymes in the cytosolic and plastidial compartments in plants; designated as PKc and PKp forms respectively (Plaxton, 1996; Givan, 1999). Transgenic tobacco plants which were deficient in PKc were used to demonstrate its role in regulating development via modulation of carbon sink-source relationships (Knowles et al., 1998; Grodzinski et al.,1999). PKc lines exhibited delayed shoot and flower development and this was correlated with poor export of previously fixed 14CO2 from leaves in the ‘night-time’ phase of a light-dark cycle but increased 14CO2 release from respiration (Grodzinski et al.,1999). Conversely, in another study with Arabidopsis seeds, PKp has been shown to play an important role in fatty acid biosynthesis (Andre and Benning, 2007; Andre et al., 2007). PKs also exist as tissue specific isozymes (Turner et al., 2005). The subtle respiratory regulation that these difference in PK isoforms affords is well-illustrated by a classic study of PKc repression and activation in castor seed endosperm (Podesta and Plaxton, 1991). In castor seeds, during aerobic conditions, the allosteric inhibition of endosperm PKc facilitated larger gluconeogenic conversion of stored triacylglycerides to hexose-phosphates assisting in germination. However, under low oxygen PKc became active in order to compensate for ATP depletion that occurs due to hypoxic stress (Podesta and Plaxton, 1991).

A key study also used a transgenic approach to provide greater insight into the role of PKc in carbon metabolism through the coordinated regulation of glycolysis, the TCA cycle, the mitochondrial ETC and also AOX in potato tuber (Oliver et al., 2008). A role for PKc in these respiratory pathways was implied from a series of observations. Firstly, pyruvate addition experiments showed an effect on glycolytic flux and the consequences that altered the dynamics of mitochondrial ETC (Zabalza et al., 2009). The link to AOX was suggested when an increase in AOX activity was seen after pyruvate was added to isolated mitochondria (Millar et al., 2003). This AOX effect was then explained through the interaction of pyruvate to cysteine residue of AOX (Umbach et al., 2006).

Transgenic potato tubers with decreased in PKc levels were generated through an RNA interference (RNAi) gene silencing approach, among which three lines were selected, lines PKC-25, 6 and 15 – where PK activity was reduced to ~40%, 37% and 29% respectively (Oliver et al., 2008). As expected, lowering PKc expression led to a higher PEP to pyruvate ratio in actively growing tubers. This decrease in pyruvate levels correlated with a decrease in the various organic acids involved in the TCA cycle and there was also a decrease in the level of total protein in the tubers. [14C]Glc labelling and feeding experiments showed a slight decrease in carbon partitioning towards organic acid and protein synthesis upon decrease in PKc levels. These results clearly demonstrated that PKc plays a very important role in the regulation of the levels of organic acids in tubers and partitioning the carbon toward the TCA cycle but interestingly total respiration and TCA cycle flux did not alter. One reason could be that residual pyruvate levels are probably enough to maintain the respiratory activity in these tubers. Equally, other enzymes that generate pyruvate such as PKp, PEPC, or PEP phosphatase could be compensating for the loss in PKc. Alternatively; there could be a compensatory change in electron transport through the COX pathway, which is in line with the electron partition model (Ribas-Carbo et al., 1995). This would imply that respiratory metabolism has a high homeostatic ability allowing considerable flexibility in response to changes in metabolite and transcript levels (Nunes-Nesi et al., 2005, 2007; Studart-Guimarães et al., 2007).

The potato RNAi lines also exhibited a suppression of AOX-dependent respiration which could be reversed by external feeding of pyruvate to tuber tissue. Suppression of the AOX pathway would be beneficial in growing tubers, which characteristically have low internal oxygen concentrations and low adenylate energy charge (Geigenberger, 2003). In line with this, PKc silenced plants produced significantly more tubers which also tended to be larger than the control tubers (Oliver et al., 2008). Thus, PKc modulation of pyruvate accumulation would be of great agronomic importance, functioning as a key regulatory step in potato tuber development by influencing the AOX in heterotrophic potato tubers.

NADPH dehydogenases linked to AOX

In addition to complex I (NADH dehydrogenase) there are some additional proteins which can use NADH and NADPH to reduce ubiquinone pool. There are NAD(P)H dehydrogenases. Type II NAD(P)H dehydrogenases (ND2) are membrane-bound proteins that face either the matrix or the inter-membrane side (Figure 1.1). Unlike complex I these are not involved in proton translocation and therefore do not contribute for ATP synthesis. As shown in Figure 1.1