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1040-9238/02/$.50© 2002 by CRC Press LLC
Critical Reviews in Biochemistry and Molecular Biology, 37(2):71–119 (2002)
Essentiality of Mitochondrial OxidativeMetabolism for Photosynthesis:Optimization of Carbon Assimilation andProtection Against Photoinhibition
K. Padmasree, L. Padmavathi, and A.S. Raghavendra*
Department of Plant Sciences, School of Life Sciences, University of Hyderabad,Hyderabad - 500 046, India
Referee: Christine Foyer, Dept. of Biochemistry and Physiology, IACR - Rothamsted,Harpemden, Herts AL5 2JQ, England, United Kingdom
* Author for correspondence.
** Name and address of corresponding author: Professor A.S. Raghavendra, Department of Plant Sciences, Schoolof Life Sciences, University of Hyderabad, Hyderabad 500 046, India. Tel: +91-40-3010630. Fax: +91-40-3010145 E-mail: [email protected]
Table of Contents
I. Introduction to the Topic .................................................... 73A. Scope of the Present Review ........................................... 74B. Differential Effects on CO2 Efflux/O2 Uptake ................. 74C. Light Enhanced Dark Respiration (LEDR)...................... 75D. Mitochondrial Respiration in Light: Modified TCA
Cycle ................................................................................. 78
II. Essentiality of Mitochondrial Respiration forPhotosynthesis...................................................................... 79A. Restriction of CO2 Assimilation, but Not of Photochemical
Activities ........................................................................... 81B. Importance at Both Limiting and Optimal CO2............... 82C. Role of Cytochrome and Alternative Pathways of
Oxidative Electron Transport ........................................... 85D. Pronounced Interaction in Algal Mutants/Guard Cells ... 86
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III. Protection Against Photoinhibition .................................... 87A. Oxidative Electron Transport and Oxidative
Phosphorylation ................................................................ 88B. Sustenance of Repair Mechanism by Mitochondrial
ATP ................................................................................... 89C. Photorespiratory Reactions ............................................... 89D. Significance Under Temperature or Water Stress ............ 91
IV. Optimization of Photosynthetic Carbon Assimilation..... 92A. Sustenance of Sucrose Biosynthesis: Role of ATP ......... 93B. Maintenance of Redox state: Ratios of malate/OAA
and triose-P/PGA.............................................................. 95C. Shortening of Induction .................................................... 97D. Activation of Enzymes ..................................................... 99E. Integration with Photorespiration and Nitrogen
Metabolism ..................................................................... 100F. Role in C4 Photosynthesis .............................................. 101
V. Biochemical Basis: Interorganelle Interaction............... 104A. Major Products of Organelle Metabolism ..................... 104B. Metabolite Exchange between Chloroplasts, Mitochondria,
Peroxisomes, and Cytosol .............................................. 106
VI. Future Perspectives........................................................... 108
ABSTRACT: The review emphasizes the essentiality of mitochondrial oxidative metabo-lism for photosynthetic carbon assimilation. Photosynthetic activity in chloroplasts andoxidative metabolism in mitochondria interact with each other and stimulate their activities.During light, the partially modified TCA cycle supplies oxoglutarate to cytosol and chlo-roplasts. The marked stimulation of O2 uptake after few minutes of photosynthetic activity,termed as light enhanced dark respiration (LEDR), is now a well-known phenomenon. Boththe cytochrome and alternative pathways of mitochondrial electron transport are importantin such interactions. The function of chloroplast is optimized by the complementary natureof mitochondrial metabolism in multiple ways: facilitation of export of excess reducedequivalents from chloroplasts, shortening of photosynthetic induction, maintenance ofphotorespiratory activity, and supply of ATP for sucrose biosynthesis as well as othercytosolic needs. Further, the mitochondrial oxidative electron transport and phosphoryla-tion also protects chloroplasts against photoinhibition. Besides mitochondrial respiration,reducing equivalents (and ATP) are used for other metabolic phenomena, such as sulfur ornitrogen metabolism and photorespiration. These reactions often involve peroxisomes and
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cytosol. The beneficial interaction between chloroplasts and mitochondria therefore extendsinvariably to also peroxisomes and cytosol. While the interorganelle exchange of metabo-lites is the known basis of such interaction, further experiments are warranted to identifyother biochemical signals between them. The uses of techniques such as on-line massspectrometric measurement, novel mutants/transgenics, and variability in metabolism bygrowth conditions hold a high promise to help the plant biologist to understand thisinteresting topic.
KEY WORDS: alternative pathway, chloroplasts, cytochrome pathway, interorganelleinteraction, mitochondria, peroxisomes.
I. INTRODUCTION TO THETOPIC
Photosynthesis, the primary source ofenergy for the living world, consists of twodistinct phases. The first phase of photo-chemical reactions, involves the conversionof radiant solar energy into chemical formslike ATP and NADPH (or reduced ferre-doxin) with concomitant evolution of oxy-gen. In the second biochemical phase, theATP and NADPH (or reduced ferredoxin)are utilized to reduce carbon dioxide (orother compounds like NO2 or SO2) intoenergy rich carbon (or nitrogen or sulfur)compounds. Respiration on the other handinvolves the oxidation of carbon compoundsand production of NADH or FADH withthe simultaneous release of CO2. The reduc-tants (NADH or FADH) are oxidizedthrough the electron transport (and oxida-tive phosphorylation) to produce ATP, in-volving consumption of O2 and release ofwater.
Thus, photosynthesis is a process ofreduction and respiration is a process ofoxidation. Both processes provide ATP forcellular needs. The nature of these twometabolic pathways implies that theycomplement each other. The major sites ofphotosynthesis and respiration are chloro-plasts and mitochondria. Although chloro-plasts and mitochondria are traditionally
considered to be autonomous organelles,recent literature has established that thesetwo organelles are not only interdependentin their functions but also are mutually ben-eficial in their interaction.
Besides the carbon metabolism, the re-duced equivalents are consumed in meta-bolic reactions of photorespiration, nitrateassimilation, and sulfur metabolism. Forexample, the requirement of NADH forhydroxypyruvate reduction in peroxisomesis met by chloroplasts as well as mitochon-dria. Naturally, the cytoplasm is a commonmedium for the flux of all related metabo-lites. Thus the interaction of chloroplastsand mitochondria is not exclusive but ex-tends to cytoplasm and peroxisomes.
Under limiting CO2, photorespiration ishighly active and becomes a major linkbetween chloroplasts, peroxisomes, cyto-plasm, and mitochondria. Glycine is themajor substrate of mitochondrial respira-tion under limiting CO2 and can contributesignificant amounts of ATP to cell. At highCO2, the enhanced requirement of ATP incytosol (for sustenance of sucrose biosyn-thesis) is met again from mitochondria(which can use both glycine and malate asrespiratory substrates). Under both situa-tions, nitrogen metabolism and recycling ofammonia/keto acids are always integratedwith the functioning of chloroplasts, mito-chondria, peroxisomes, and cytoplasm. Anymodulation of respiration leads to changes
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in the patterns of photosynthesis and photo-respiration and subsequently modificationof nitrogen as well as sulfur metabolism.
The current review attempts to criticallyassess and emphasize the physiological andbiochemical features of interorganelle inter-action: chloroplasts, mitochondria, peroxi-somes, and cytoplasm. However, emphasisis given to the essentiality of mitochondrialrespiration for photosynthetic carbon metabo-lism. The mitochondrial oxidative metabo-lism not only helps to optimize photosynthe-sis in varied environmental conditions butalso protects the chloroplasts against thephotoinhibition of photosynthesis.
A. Scope of the Present Review
Due to the intriguing but interestingnature of the topic, considerable effort hasbeen made in the last decade to study theoccurrence of mitochondrial respiration inlight and its importance for photosynthesis,particularly in mesophyll protoplasts andleaves. In view of the limited space, all theoriginal articles are not referred to in thisreview. Readers interested in the extensiveliterature in this and related areas may con-sult the previous reviews (Azcón-Bieto,1992; Raghavendra et al., 1994; Krömer,1995; Gardeström and Lernmark, 1995;Gardeström, 1996; Hoefnagel et al., 1998;Padmasree and Raghavendra, 1998, 2000;Atkin et al., 2000b; Gardeström et al., 2002).
Most of the work on the interaction ofmitochondrial respiration and chloroplastphotosynthesis is based on the use of meta-bolic inhibitors that inhibit specific reac-tions at low concentrations. An inherentdisadvantage, however, is that these inhibi-tors cause nonspecific effects particularly athigher concentrations (see Section VI).Among the mitochondrial inhibitors referredfrequently in this review are rotenone (an
inhibitor of complex I in the mitochondrialrespiratory chain); antimycin A (an inhibi-tor of complex III); KCN/NaN3 (inhibitorsof complex IV); oligomycin (an inhibitor ofcomplex V); salicylhydroxamic acid(SHAM)/propyl gallate (inhibitors of alter-native oxidase); and aminoacetonitrile(AAN, an inhibitor of glycine decarboxy-lase).
B. Differential Effects on CO 2
Efflux/O 2 Uptake
The occurrence of mitochondrial respi-ration in light has been a matter of debate, fora long time, because of ambiguous reports onthe extent and pattern of dark respiration inlight (Graham, 1980; Raghavendra et al.,1994; Krömer, 1995; Villar et al., 1995; Atkinet al., 1997; Hoefnagel et al., 1998; Atkin etal., 2000a). Some studies have indicated thatdark respiration was either unaffected orstimulated, while others found that respira-tion was inhibited (Table 1). Such a largevariation in these reports appears to be due toa combination of factors: the component ofdark respiration being monitored (CO2 efflux/O2 uptake), the experimental technique be-ing used, and the subject of experimentalsystem (leaves, algal cells, or cell cultures).
It is difficult to monitor precisely CO2/O2 exchange in light by conventional meth-ods because the measurements are compro-mised by the occurrence of related phenom-ena besides dark respiration, for example,photorespiration, photosynthesis, Mehlerreaction, chlororespiration. Each one of theabove processes contributes significantly tothe net CO2 or O2 exchange.
A promising solution was provided bythe technique of mass spectrometry, whichcould distinguish between the uptake/effluxof O2 or CO2 occurring simultaneously dur-ing respiration, photosynthesis, and related
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cellular processes. Mass spectrometric stud-ies using 13/12CO2 and 18/16O2 have revealedthat light has a differential effect on CO2
efflux and O2 uptake (Avelange et al., 1991;Raghavendra et al., 1994; Xue et al., 1996;Atkin et al., 2000a). On illumination, CO2
efflux is suppressed by almost 81%, whileO2 consumption is either unaffected or stimu-lated up to 3.5-fold (Table 2). A major ob-servation from these mass spectrometric ex-periments is that mitochondrial oxidativeelectron transport continues to be active,
irrespective of illumination. The sustenanceof active mitochondrial oxidative electrontransport is essential for optimal photosyn-thesis.
C. Light-Enhanced DarkRespiration (LEDR)
Although the extent of respiration in lightis often debated, the stimulation of dark res-
TABLE 1The Effect of Light on Dark Respiration in Plant Tissues, Determined by DifferentTechniques, Indicating a Large Variation in the Extent of Inhibition
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piration due to illumination, particularly ingreen tissues, is now well established. Therespiratory O2 uptake in dark increases quitesignificantly, soon after illumination (Figure 1).This phenomenon termed as ‘light enhanceddark respiration’ (LEDR) occurs after evenshort periods of exposure to light (Padmasreeand Raghavendra, 1998). The phenomenonof LEDR has been recorded in different ex-perimental systems and the extent of stimu-lation by light, varied from 1.2- to 7-fold(Table 3).
The extent of LEDR is positively corre-lated with the intensity and duration of thepreceeding period of illumination (Raghavendraet al., 1994; Xue et al., 1996). The sensitivity ofLEDR to DCMU (an inhibitor of photosystemII electron transport) and D,L-glyceraldehyde
(inhibitor of Calvin cycle) establishes that LEDRis dependent on products of photosyntheticcarbon assimilation and electron transport(Reddy et al., 1991). Exposure of Euglena gra-cilis, a flagellate to UV radiation decreasedboth the rate of photosynthesis and LEDR,especially at higher light intensities (Ekelund,2000).
LEDR is different from photorespiratorypost-illumination burst (PIB). The phenom-enon of PIB results due to CO2 released dur-ing decarboxylation of photorespiratory gly-cine. In tobacco leaf PIB occurs within 20 safter the light is switched off, while LEDRoccurs between 180 to 250 s after stoppingillumination (Atkin et al., 1998). Further at2% O2 (where photorespiration is minimized)PIB is not seen, whereas LEDR is still ob-
TABLE 2Selected Examples Showing the Differential Effect of Light on Respiratory CO 2 Releaseand O2 Uptake, as Determined by Mass Spectrometry. On Illumination, the DecarboxylationReactions Are Usually Inhibited Resulting in a Decrease of CO 2 Release, While the Processof Oxidative Electron Transport (Indicated by O 2 Uptake) Is Either Unaffected or EvenEnhanced
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served (Atkin et al., 1998; Atkin et al., 2000a).LEDR is also insensitive to AAN (an inhibi-tor of mitochondrial glycine metabolism)demonstrating that LEDR is not directly re-lated to photorespiration (Gardeström et al.,1992; Raghavendra et al., 1994).
The occurrence of LEDR within a fewminutes suggests that the interaction be-tween photosynthesis and respiration is quiterapid and involves primary photosyntheticproducts, especially malate (Raghavendraet al., 1994; Padmasree and Raghavendra,1998). The malate concentration is high atthe end of illumination, and it is rapidlymetabolized during the subsequent darkperiod (Hill and Bryce, 1992). In contrast,
the levels of sucrose, glucose, and fructosedid not change significantly during LEDRin the mesophyll protoplasts of barley (Hilland Bryce, 1992).
Plant mitochondrial pyruvate dehydro-genase complex (PDC) and NAD-malicenzyme are reversibly inhibited in light(see Section I.D). On switching over todarkness, these two enzymes are reacti-vated. Photosynthetically generated malateis oxidized via both malate dehydroge-nase (MDH) and NAD-malic enzyme,resulting in the formation of oxaloacetate(OAA) or pyruvate and CO2. Pyruvate isdecarboxylated by PDC and converted intoacetyl CoA. Both OAA and acetyl CoA
FIGURE 1. LEDR in mesophyll protoplasts of pea. The rate of respirationwas stimulated by three-fold after 15 min of illumination, but not whenprotoplasts were kept in darkness for similar periods. (Modified fromReddy et al., 1991.)
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enter TCA cycle and enhance the processof respiratory CO2 release and oxidativeelectron transport. All these result inan upsurge in CO2 release or O2 uptaketermed LEDR (Raghavendra et al., 1994;Padmasree and Raghavendra, 1998; Atkinet al., 2000a). Thus, malate could be thesubstrate and signal for LEDR, whichcould accomplish the rapid conversion ofphotosynthetically generated reducingpower into ATP. Experiments designed toalter chloroplast malate production or theimport of malate into mitochondria, forinstance, by altering the function of chlo-roplastic MDH or the overexpression/supression of mitochondrial OAA/malatetranslocator would help throw more lighton the cause of LEDR.
D. Mitochondrial Respiration inLight: Modified TCA Cycle
Dark respiration consists of three steps:(1) glycolysis in the cytosol, (2) the TCAcycle, consisting of decarboxylation of car-bon compounds resulting in the productionof NADH/FADH and CO2, (3) the electrontransport chain involving NADH/FADHoxidation to produce ATP, and O2 consump-tion.
The processes of CO2 production andO2 uptake are not as tightly coupled in lightas in darkness. As discussed in the previoussection, there is usually a reduction in theextent of respiratory CO2 efflux, while oxy-gen uptake is either unaffected or even stimu-lated (Table 2). The main reason for such
TABLE 3The Occurrence and Extent of Light Enhanced Dark Respiration (LEDR) in Plant Tissues.The Rate of Respiration Increases Markedly After a Few Minutes of PhotosyntheticCarbon Assimilation and LEDR Is Represented as the % Increase in Respiration JustAfter Illumination Over the Rate of Respiration (Steady State) in Continuous Darkness
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decrease in CO2 release is the partial inhibi-tion/modification of TCA cycle activityoccurring in light. Two of the possible causesthat downregulate/modify TCA cycle activ-ity are (1) reversible inactivation of mito-chondrial pyruvate dehydrogenase complexin light (Budde and Randall, 1990), (2) rapidexport of oxoglutarate out of mitochondria(Hanning and Heldt, 1993), thus resultingin a short-circuit of Kreb’s cycle.
PDC is phosphorylated (inactivated) inlight by a PDC-protein kinase and dephos-phorylated (activated) in darkness (Leuthyet al., 1996; Randall et al. 1996). The inac-tivation of PDC is linked to photosyntheticactivity as indicated by its sensitivity toDCMU (inhibitor of PSII) and the absenceof the phenomenon in etiolated seedlings.Further, the products of glycine decarboxy-lation, NADH and NH4
+, also enhance thephosphorylation of PDC. Conditions thatreduce photorespiration (high CO2 and/orlow O2) limit the extent of PDC inactiva-tion. Taken together these reports indicatethat under conditions of high rates of photo-synthesis or photorespiration, PDC is inac-tivated, but oxidation of glycine or malatecontinues.
The second possible reason for reducedCO2 efflux in light is the export of TCAcycle compounds from mitochondria tochloroplast (mainly for NH4+ assimilationin light), thus limiting the substrates avail-able for further steps of the TCA cycle.Mitochondria export TCA cycle interme-diates in the form of citrate, which is con-verted into oxoglutarate in cytosol and sentinto chloroplast (Figure 2; Chen and Gadal,1990; Gout et al., 1993; Hanning and Heldt,1993; Krömer, 1995; Atkin et al., 2000b).This results in a partial activity of TCAcycle in light resulting in reduced CO2
efflux (Parnik and Keerberg, 1995). Thusthe main function of TCA cycle in lightseems to be the supply of carbon skeletonsto the chloroplasts for ammonium assimi-
lation (Weger et al., 1988 and Weger andTurpin, 1989).
The ATP levels in cytosol during lightdo not appear to play any crucial role indownregulating TCA cycle. The ATP/ADPratio required in the cytosol to decreasemitochondrial respiration is much higherthan that usually occurs. In fact, the ATP/ADP ratio in cytosol is lower in light com-pared to darkness at saturating CO2 levels(Krömer, 1995; Atkin et al., 2000b). If mi-tochondrial function in light were to be in-hibited due to high ATP levels in cytosol, itwould have also inhibited O2 uptake besidesCO2 release. However, experimental evi-dence shows that O2 uptake does not de-crease much in light. The mitochondrialelectron transport chain oxidizes not onlythe NADH produced by the partially activeTCA cycle, but also that produced byphotorespiratory glycine decarboxylation.Glycine oxidation therefore can contributesignificantly to oxygen consumption bymitochondria in light.
II. ESSENTIALITY OFMITOCHONDRIAL RESPIRATIONFOR PHOTOSYNTHESIS
One of the first indications about theimportance of mitochondria came from thefrequent observation of a positive relation-ship between dark respiration and photosyn-thesis. A strong positive correlation existsbetween steady state rates of photosynthesisand respiration in a wide range of species(Ceulemans and Saugier, 1991) and the res-piratory rate of leaves increases significantlyafter the light period or hours of illumination(Raghavendra et al., 1994; Atkin et al., 1998).Mitochondrial oxidative metabolism (particu-larly oxidative electron transport and oxida-tive phosphorylation) has been shown to beessential for maintaining high rates of photo-
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FIG
UR
E 2
. Dur
ing
illum
inat
ion
a pa
rtia
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TC
A c
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s in
mito
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thet
ic c
ells
. A
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part
of O
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is c
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, de
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to
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to b
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oxog
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o be
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ly t
o cy
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l. H
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er,
an N
AD
P d
epen
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IC
DH
ope
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s w
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e is
con
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ed t
o 2-
oxog
luta
rate
. T
he o
ther
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ctio
ns o
fT
CA
cyc
le,
whi
ch a
re r
estr
icte
d in
lig
ht a
re i
ndic
ated
by
dash
ed a
rrow
. T
he i
mpo
rtan
t en
zym
es i
nvol
ved
are
PE
PC
, ph
osph
oeno
lpyr
uvat
eca
rbox
ylas
e; P
K, p
yruv
ate
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se; M
DH
, mal
ate
dehy
drog
enas
e; M
E, m
alic
enz
yme;
PD
C, p
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ate
dehy
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enas
e co
mpl
ex; C
S, c
itrat
e sy
ntha
se;
AC
N,
cis-
acon
itase
; ID
H,
isoc
itrat
e de
hydr
ogen
ase.
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synthesis under a variety of conditions. Thebeneficial effects of mitochondria are knownduring not only short cycles of darkness andillumination, but also under stress environ-ments like photoinhibitory light or low tem-perature (Vani et al., 1990; Saradadevi andRaghavendra 1992; Saradadevi et al., 1992;Shyam et al., 1993; Hurry et al., 1995).
The marked interaction between photo-synthesis and respiration is convincinglydemonstrated by experiments with leaf me-sophyll protoplasts and intact leaves (Krömeret al., 1988; Vani et al., 1990; Krömer andHedlt, 1991a; Krömer et al., 1993; Padmasreeand Raghavendra, 1999a,b,c) using inhibi-tors of mitochondrial metabolism. The es-sentiality of different components of mito-chondrial respiration for chloroplasticphotosynthesis is documented by the use oftypical mitochondrial inhibitors. For example,oligomycin was employed as inhibitor ofoxidative phosphorylation (Krömer et al.,1988; Krömer and Heldt, 1991a; Krömer etal., 1993), antimycin A to inhibit the cyto-chrome pathway (Igamberdiev et al., 1997a,b;Padmasree and Raghavendra, 1999a,b,c),while SHAM was used to inhibit the alterna-tive pathway (Igamberdiev et al., 1997a,b;Padmasree and Raghavendra, 1999a,b,c). Onthe other hand, the importance of glycolyticreactions and TCA cycle in stimulating pho-tosynthetic O2 evolution was assessed by theusage of sodium fluoride and sodium mal-onate, respectively (Vani et al., 1990). Re-cently, studies were carried out using mu-tants deficient in mitochondrial glycinedecarboxylase (Igamberdiev et al., 2001).
A. Restriction of CO 2
Assimilation, but Not ofPhotochemical Activities
Preliminary experiments have indicatedthat the inhibitors employed during these
studies had no direct effect on chloroplasts(Krömer et al., 1988; Padmasree andRaghavendra, 1999a). The suppression ofphotosynthetic activity was reversed and thefull rate was restored when the protoplastswere ruptured, leaving the chloroplasts in-tact. These results indicate that the stronginhibition of photosynthesis observed witholigomycin or antimycin A or SHAM wasdue to not an effect on chloroplast photo-synthesis as such, but interference of reac-tions between the chloroplasts, cytosol andmitochondria (Figure 3).
The statistical significance of the inter-action between photosynthesis and respira-tion only in the presence of CO2 (but not inits absence) suggested that carbon assimila-tion was a prerequisite (Vani et al., 1990). Itis intriguing to note that despite the smalleffects of SHAM on respiration or ATPlevels, the decrease in photosynthetic activ-ity is always pronounced (Padmasree andRaghavendra, 1999a).
A recent comprehensive study reexam-ined the effects of mitochondrial inhibitors:oligomycin, antimycin A, and SHAM onthe photosynthetic carbon assimilation andphotochemical electron transport activities,monitored in intact mesophyll protoplasts(Padmasree and Raghavendra, 2001b).When mesophyll protoplasts were illumi-nated in presence of mitochondrial inhibi-tors, there was a significant decrease (>45%)in HCO3
–-dependent O2 evolution, while thedecrease in O2 evolution was marginal(<10%) in presence of benzoquinone (BQ),[PSII mediated] and NO2– [dependent onPSII + PSI] as electron acceptors (Figure 4).DCMU, a typical photosynthetic inhibitordecreased drastically all the three reactions:HCO3
– or BQ or NO2–-dependent O2 evolu-
tion in mesophyll protoplasts. The effect ofmitochondrial inhibitors on photosyntheticreactions was similar in the presence orabsence of NH4Cl, an uncoupler, indicatingthat photophosphorylation also was not af-
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82
fected. Thus, mitochondrial oxidative me-tabolism (through both cytochrome and al-ternative pathways) was essential for themaintenance of photosynthetic carbon as-similation, but had no direct effect on PSI-or PSII-dependent photochemical electrontransport activities in mesophyll protoplastsof pea. However, during a long-term incu-bation, interference with mitochondrial me-tabolism can lead to disturbance in photo-chemical activities through feedback effectsof thylakoid overenergization (Krömer etal., 1993).
B. Importance at Both Limitingand Optimal CO 2
The rate of photosynthetic O2 evolutiondepends on not only light intensity but alsothe CO2 concentration. At saturating CO2
and light, the rate of photosynthesis is lim-ited by the flux of assimilated carbon intosucrose and at limiting CO2 and saturatinglight, the rate of photosynthesis is limited
by rubisco activity (Krömer et al., 1993). Atoptimal CO2 (nonphotorespiratory condi-tions), the photosynthetic demand for ATPis expected to be very high, while such aneed for ATP would be lower at limitingCO2. The decrease in the rate of photosyn-thesis due to mitochondrial inhibitors, oli-gomycin, antimycin A, or SHAM at opti-mal CO2 (1.0 mM NaHCO3) was muchstronger than that at limiting CO2 (0.1 mMNaHCO3) under similar conditions (Figure 5).Nevertheless, the significant decrease in therate of photosynthesis under both limitingand optimal CO2 in the presence of theseinhibitors suggests that mitochondrial oxi-dative metabolism is essential for maximalphotosynthesis at both limiting CO2
(photorespiratory conditions) as well asoptimal CO2 (Padmasree and Raghavendra,1999a).
At optimal CO2, most of the photosyn-thate is converted to sucrose-consuming ATPand Glc-6-P. Both oligomycin and antimycinA while causing a decrease in photosynthesisalso raised the levels of Glc-6-P and triose-Pat optimal CO2 (Krömer et al., 1988; Krömer
FIGURE 3. Marked suppression of bicarbonate dependent O2 evolution (�) in pea mesophyllprotoplasts by both antimycin A (inhibitor of cytochrome pathway of mitochondrial electron trans-port) and SHAM (inhibitor of alternative pathway). These two compounds had no direct effect onchloroplast photosynthesis (∆). (Adapted from Padmasree and Raghavendra, 1999a.)
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FIG
UR
E 4
. C
hang
e in
rat
es o
f ox
ygen
evo
lutio
n w
hen
mes
ophy
ll pr
otop
last
s ar
e in
cuba
ted
with
diff
eren
tco
ncen
trat
ions
of
olig
omyc
in,
antim
ycin
A,
DC
MU
or
SH
AM
. �
: H
CO
3–-d
epen
dent
O2
evol
utio
n (r
efle
cts
the
tren
d of
CO
2 a
ssim
ilatio
n);
�:
Rat
es o
f ox
ygen
evo
lutio
n w
hen
elec
tron
s ar
e tr
ansf
erre
d fr
om H
2O
to
NO
2
(rep
rese
ntin
g P
SII
and
PS
I ac
tivity
); ∆
: B
enzo
quin
one-
depe
nden
t ox
ygen
evo
lutio
n (in
dica
tes
PS
II ac
tivity
excl
udin
g P
SI,
H2O
, to
BQ
). In
con
trol
s, th
e ra
tes
of O
2 ev
olut
ion
in µ
mol
mg–1
Chl
h–1
und
er d
iffer
ent c
ondi
tions
wer
e 15
4 (H
CO
3– -
depe
nden
t O
2 ev
olut
ion)
; 44
5 (B
Q-d
epen
dent
O2
evol
utio
n) o
r 52
(N
O2-d
epen
dent
O2
evol
utio
n).
(Ada
pted
fro
m P
adm
asre
e an
d R
agha
vend
ra,
2001
b.)
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FIG
UR
E 5
. E
ffect
of
antim
ycin
A (
A)
or S
HA
M (
B)
on p
hoto
synt
hesi
s at
opt
imal
(1.
0 m
M N
aHC
O3)
or li
miti
ng (
0.1
mM
NaH
CO
3)
CO
2 in
mes
ophy
ll pr
otop
last
s of
pea
. (M
odifi
ed f
rom
Pad
mas
ree
and
Rag
have
ndra
, 19
99a.
)
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and Heldt, 1991a; Krömer et al., 1993;Padmasree and Raghavendra, 1999a). Obvi-ously, mitochondrial metabolism and ATPsupply are essential for the maintenance ofsucrose biosynthesis. At limiting CO2 there iseither no change or only a marginal decreasein Glc-6-P in the presence of oligomycin orantimycin A (Padmasree and Raghavendra,1999a). However, the presence of SHAM(which leads to only a limited ATP produc-tion) caused a decrease in the levels of Glc-6-Pand had no effect on ATP/ADP levels,while markedly affecting photosynthesis inmesophyll protoplasts (Padmasree andRaghavendra, 1999a). Thus, the mitochon-drial supply of ATP for sucrose synthesis maybe only a secondary factor during the interac-tion with photosynthesis at limiting CO2.
Although the marked decrease in the rateof photosynthetic O2 evolution at both opti-mal and limiting CO2 demonstrates the es-sentiality of mitochondrial oxidative metabo-lism in optimizing photosynthesis, underphotorespiratory conditions mitochondrialelectron transport is more crucial than oxida-tive phosphorylation in benefitting photosyn-thesis. A major function of the mitochon-drion in a photosynthesizing cell, particularlyunder low light intensities and optimal CO2,seems to be the supply of ATP for cytosoliccarbon metabolism, that is, sucrose synthe-sis. In high light, mitochondria take on theadditional role of oxidizing the excess reduc-ing equivalents generated by photosynthesis,preventing overreduction of chloroplasticredox carriers and thus maintaining high ratesof photosynthesis (Krömer, 1995; Padmasreeand Raghavendra, 1998, 2000).
C. Role of Cytochrome andAlternative Pathways ofOxidative Electron Transport
The sensitivity of protoplast photosyn-thesis to mitochondrial inhibitors at limiting
CO2 (when the ATP requirement for photo-synthesis is expected to be low) and the lackof correlation between the photosyntheticrates and the ratios of ATP/ADP in proto-plasts (particularly in presence of SHAM)have indicated that mitochondrial electrontransport activity is more important than theoxidative phosphorylation for optimal pho-tosynthesis (Padmasree and Raghavendra,1999a).
Plant mitochondria have the unique ca-pability of oxidizing NADH/FADH throughtheir electron transport by two differentroutes: (1) cyanide-sensitive cytochromepathway and (2) cyanide-resistant alterna-tive pathway (Lambers, 1985; Vanlerbergheand McIntosh, 1997; Mackenzie and McIn-tosh, 1999). The alternative pathway is cata-lyzed by alternative oxidase (AOX), whichhas been purified, characterized, and its geneisolated (Siedow and Umbach, 2000). Whilethe molecular biology and regulation ofAOX are studied in detail (McIntosh, 1994;Siedow and Umbach, 1995, 2000), the in-formation on the physiological significance/metabolic function of AOX is still limited.
The relative proportion of cytochromeand alternative pathways is flexible andvaries with environmental conditions suchas temperature, age of the tissue, and injury/wounding. The circumstantial evidence sug-gests that the operation of alternative path-way is likely to increase in illuminated planttissues. The levels of AOX increase duringgreening of etiolated leaves (Atkin et al.,1993). The accumulation of sugars duringthe illumination promotes the engagementof alternative pathway (Azcón-Bieto, 1992).A significant part of the light-enhanced darkrespiration (LEDR) appears to involve al-ternative pathway (Igamberdiev et al.,1997a). It is not known, however, if there isany modulation by illumination of the ex-tent of mitochondrial electron transportthrough the alternative pathway.
Although the essentiality of mitochon-drial oxidative phosphorylation for photo-
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synthetic carbon assimilation is well estab-lished, the role of cytochrome and alterna-tive pathways in benefitting photosyntheticmetabolism is examined only to a limitedextent. The importance of cytochrome andalternative pathways during photosynthesiswas studied in mesophyll protoplasts of peaand barley, using low concentrationsof mitochondrial inhibitors: oligomycin (in-hibitor of oxidative phosphorylation), anti-mycin A (inhibitor of cytochrome pathway)and salicylhydroxamic acid (SHAM, an in-hibitor of alternative pathway). All thethree compounds decreased the rate of pho-tosynthetic O2 evolution in mesophyll pro-toplasts, but did not affect chloroplast pho-tosynthesis (Krömer et al., 1988; Krömerand Heldt, 1991a; Krömer et al., 1993;Igamberdiev et al., 1997a, 1998; Padmasreeand Raghavendra, 1999a,b,c). The markedsensitivity of photosynthesis to both SHAMand antimycin A suggests that the alterna-tive pathway is as essential as the cyto-chrome pathway for optimal photosynthe-sis. These results also demonstrate animportant role of the alternative pathway inplant cells: essentiality for chloroplast pho-tosynthesis.
The importance of the alternative path-way during the interaction between respira-tion and photosynthesis is suggested by alsothe sensitivity of LEDR to SHAM in meso-phyll protoplasts of barley (Igamberdiev etal., 1997a) and algae Selenastum minutum,Chlamydomonas reinhardtii, and Euglenagracilis (Lynnes and Weger, 1996; Xue etal., 1996; Ekelund, 2000).
Restriction of cytochrome pathway byantimycin A or uncoupling of cytochromepathway from oxidative phosphorylation byoligomycin prolonged both the inductionphase of photosynthesis and the activationof NADP-MDH during transition from darkto light (Igamberdiev et al., 1998; Padmasreeand Raghavendra, 1999b). The exact mecha-nism of the optimization of photosynthesis
by alternative pathway is not completelyunderstood, but one of the reasons appearsto be the effective modulation of intracellu-lar redox state (Padmasree and Raghavendra,1999c). A major function of AOX pathwayin mesophyll cells can be the maintainanceof the oxidation of malate, particularly un-der excess light (see Section IV.B).
A recent report suggests that the phe-nomenon of the Kok effect (progressive lightinduced inhibition of dark respiration at lowlight intensities) is modulated strongly bycytochrome pathway of mitochondrial elec-tron transport. The alternative pathway ap-pears to be less important in modulating theKok effect (Padmavathi and Raghavendra,2001).
D. Pronounced Interaction inAlgal Mutants/Guard Cells
The interaction between respiration andphotosynthesis is quite pronounced in cellsthat are deficient in Rubisco/Calvin cycleactivity, such as stomatal guard cells andmutants of Chlamydomonas (Raghavendraet al., 1994).
Guard cells have high rates of respiratoryactivity but contain very low levels of Rubiscoand consequently limited carbon metabolismthrough Calvin cycle (Raghavendra and Vani,1989; Parvathi and Raghavendra, 1995).Despite the limited CO2 fixation in guardcells, the reduced equivalents produced bytheir chloroplasts are exported to the cytosolthrough OAA-malate or PGA-DHAP shuttles(Shimazaki et al., 1989). The reduced pyri-dine nucleotides formed in the cytosol fromthe oxidation of malate and/or DHAP mayact as the respiratory substrates for mito-chondrial ATP production needed for K+
uptake. A very strong interaction betweenrespiration and photosynthesis has beenshown in guard cell protoplasts of Vicia faba
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and Brassica napus at varying O2 concentra-tions (Mawson, 1993). A strong cooperationbetween chloroplasts and mitochondria ap-pears to be essential for the maintenance ofguard-cell bioenergetic processes.
A similar situation appears to operate intwo mutants of Chlamydomonas reinhardtii,one devoid of Rubisco and the other lackingfunctional chloroplast ATP synthase. TheC. reinhardtii mutant FUD50 lacks theβ-subunit of chloroplast ATP synthase andcannot produce ATP during photophospho-rylation (Gans and Rébéille, 1988). A modi-fied strain of this mutant FUD50su can growunder photoautotrophic conditions, althoughit still showed no synthesis of the β-subunitof the coupling factor. Photosynthesis inFUD50su mutant was extremely sensitiveto inhibitor antimycin A, a specific inhibitorof mitochondrial electron transport. Photo-synthesis in the FUD50su strain is achievedthrough an unusual interaction betweenmitochondria and chloroplasts (Lemaire etal., 1988). The export of reduced com-pounds, made in light, from the chloroplastto the mitochondria elicits ATP formationin the latter, and ATP is subsequently im-ported to the chloroplast.
III. PROTECTION AGAINSTPHOTOINHIBITION
Photoinhibition can be defined as themarked decrease in the photosynthetic rateunder supraoptimal light or limitation onCO2 assimilation. Such situations developwhen there is excess light or conditions lim-iting the biochemical reactions, for example,low temperature, water stress, limiting CO2,limiting N2, or any limitation on enzymes.The function of photochemical electrontransport is optimized when the reductantsgenerated in light are quickly used up forbiochemical reduction of carbon (or nitro-
gen or sulfur). Any imbalance between thephotochemical and biochemical processesleads to the phenomenon of photoinhibition(Long et al., 1994; Andersson and Barber,1996).
Photoinhibition occurs due to theoverreduction of the photosynthetic elec-tron transport system. Reactive oxygen spe-cies generated in the light as a result of theMehler reaction can lead to damage of thephotochemical apparatus, particularly PS II.The chloroplasts have the necessary ma-chinery to repair the damage caused to PSII(Carpentier, 1997; Critchley, 1998; Ohad etal., 2000). The recovery is accomplished bya continuous synthesis of PSII components,particularly D1 protein. However, such re-covery is optimal under very low light andis rather slow at moderate light intensities(Park et al., 1996; Singh et al., 1996; Ander-son, 2001). Thus, photoinhibition of photo-synthesis sets in when the rate of damageexceeds that of repair (Long et al., 1994;Critchley, 1998).
Plants have evolved in different ways tocope with photoinhibition by preventive aswell as repair mechanisms. Some of themare adjustment of chloroplast antennae size,xanthophyll cycle, CO2 fixation, photores-piration, water-water cycle, PS I cyclic elec-tron transport, scavenging reactive moleculesthrough antioxidant enzymes, rapid turn-over of D1 protein of PS II (Niyogi, 1999).However, in the past decade there has beenconvincing evidence to show that mitochon-drial respiration, especially oxidative elec-tron transport and phosphorylation, playa significant role in protecting the chloro-plasts from photoinhibition (Saradadevi andRaghavendra, 1992; Shyam et al., 1993;Raghavendra et al., 1994; Singh et al.,1996; Padmasree and Raghavendra, 1998;Atkin et al., 2000b). The protection of chlo-roplast photosynthetic machinery againstphotoinhibition is accomplished by mito-chondria through not only the oxidative elec-
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tron transport/oxidative phosphorylation butalso through the key photorespiratory reac-tions.
A. Oxidative Electron Transportand Oxidative Phosphorylation
Even at very low concentrations, anti-mycin A or sodium azide or oligomycin en-hanced markedly the extent of photoinhibitionin mesophyll protoplasts of pea (Saradadeviand Raghavendra, 1992). These inhibitiors atsuch low concentrations did not affect photo-synthesis directly. Sodium fluoride (inhibitorof glycolysis) or sodium malonate (inhibitorof TCA cycle) did not significantly affectphotoinhibition (Table 4). Apparently, oxi-dative electron transport and phosphoryla-
tion play a major role in protecting photosyn-thesis aganist photoinhibition.
After an initial increase, dark respirationdecreases significantly after prolonged expo-sure to photoinhibitory light in pea mesophyllprotoplasts (Saradadevi and Raghavendra,1992) or algal cells of Anacystis nidulans andChlamydomonas reinhardtii (Shyam et al.,1993; Singh et al., 1996). These observationsindicate a marked correlation between chloro-plast and mitochondrial activity during evenphotoinhibition. The initial increase mightrepresent an enhanced oxidation of excessredox equivalents generated by chloroplastunder high light. A subsequent decrease isprobably due to the reduced flux of redoxequivalents from chloroplasts, which are nowphotoinhibited.
The initial increase in respiration oc-curred even in the presence of KCN in
TABLE 4A Comparison of the Effect of Five Metabolic Inhibitors on Respiration, Photosynthesis,and Photoinhibition in Mesophyll Protoplasts of Pea. The Protoplasts Were Examined forTheir Photosynthetic Activity After a Preincubation (With or Without Inhibitors) for 10min at 30 oC in Either Darkness or Photoinhibitory Light (Adapted from Saradadevi andRaghavendra, 1992)
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Chlamydomonas reinhardtii (Singh et al.,1996), implying that it mostly representsthe activity of the alternative pathway (Singhet al., 1996). The alternative pathway ofmitochondrial oxidative electron transportis a potential channel for “overflow” anddissipation of excess photosynthetic reduc-tants (Lambers, 1982; Millar and Day, 1997).Alternative pathway therefore is likely toplay a significant part in preventing theoverreduction of chloroplast photosyntheticapparatus and to alleviate photoinhibition.Further experiments are needed to confirmthe role of the alternative pathway of mito-chondrial oxidative electron transport inprotection aganist photoinhibition.
B. Sustenance of RepairMechanism by MitochondrialATP
Photoinhibition is often a result ofimbalance between the synthesis and deg-radation of D1 protein. Supraoptimal lightaccelerates the degradation while slow-ing down the process of synthesis of D1protein and subsequent recovery (Figure 6).In the cyanobacterium Anacystis nidulansand green alga Chlamydomonas rein-hardtii, inhibition of dark respiration byNaN3 or KCN not only increased photo-inhibition but also accelerated photoin-hibition (Shyam et al., 1993; Singh et al.,1996). The uncoupler FCCP also had asimilar effect of intensifiyng and hasten-ing photoinhibition in both the organ-isms (Shyam et al., 1993; Singh et al.,1996).
In algal cells, mitochondrial respirationmay help in even the recovery of photosyn-thesis after photoinhibition. Treatment withsodium azide or FCCP slowed down recov-ery in Anacystis nidulans (Shyam et al.,1993). Similarly, the use of KCN and FCCP
impaired the reactivation of photosynthesisin Chlamydomonas reinhardtii (Singh et al.,1996). The above results imply that the pro-cess of recovery that involves synthesis ofD1 protein is helped by mitochondrial oxi-dative phosphorylation (Singh et al., 1996).
C. Photorespiratory Reactions
In C3 plants, photorespiration helps inreducing/preventing the damage caused bysupraoptimal light. A classic and convinc-ing demonstration of such a role is providedby transgenic tobacco plants with alteredGS activity. The transgenics with increasedGS2 (a key photorespiratory enzyme) activ-ity had higher photorespiratory rates andmore tolerance to supraoptimal light thanthe wild-type plants. On the other hand,those with reduced GS2 were low on photo-respiration and were sensitive to high light(Kozaki and Takeba, 1996).
There are two possible ways by whichphotorespiration could help preventphotoinhibition under excess light and lim-ited CO2 assimilation: (1) by using up thereducing power generated by photochemi-cal reactions in chloroplasts and (2) main-taining the optimal Pi levels in chloroplasts.When stomata are closed (e.g., drought) orwhen CO2 is limiting, disturbance in thelevels of NADPH and ATP is prevented byregulatory mechanisms, which include pho-tosynthetic control and photorespiratoryglycolate metabolism (Osmond et al.,1997). Photorespiration was shown to beessential and even more important than theMehler or Asada reactions in preventingphotoinactivation of photosynthesis in Che-nopodium bonus-henricus (Heber et al.,1996).
When protoplasts of barley mutants withreduced glycine decarboxylation were incu-bated in limiting CO2, their chloroplasts had
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FIG
UR
E 6
. Im
port
ance
of m
itoch
ondr
ial o
xida
tive
elec
tron
tran
spor
t for
pho
tosy
nthe
sis
durin
g ph
otoi
nhib
ition
or r
ecov
ery
afte
r ph
otoi
nhib
ition
in A
nacy
stis
nid
ulan
s. (
A)
Effe
ct o
f pho
toin
hibi
tory
ligh
t (25
00 µ
mol
m–2
s–1
) on
pho
tosy
nthe
sis
in th
eab
senc
e (∆
) or
pre
senc
e (∆
) of
1 m
M s
odiu
m a
zide
. (B
) R
eact
ivat
ion
of p
hoto
synt
hesi
s fr
om p
hoto
inhi
bitio
n in
the
abse
nce
or p
rese
nce
of 1
mM
sod
ium
azi
de.
50%
pho
toin
hibi
ted
cultu
res
wer
e us
ed t
o st
udy
the
reac
tivat
ion
ofph
otos
ynth
esis
fro
m u
nder
dim
ligh
t. (M
odifi
ed f
rom
Shy
am e
t al
., 19
93.)
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high ratios of ATP/ADP and NADPH/NADP (Igamberdiev et al., 2001). This in-dicates that glycine decarboxylation andassociated NH+4 recycling are sinks for ex-cess chloroplastic reductants and help toprevent a overreduction of the chloroplast.The GDC-deficient barley mutants alsoshowed significant increase in the activityof malate valve and chloroplastic NADP-MDH, apart from an increase in the activityof NAD-MDH of cytosol and mitochon-dria. Obviously, the malate shuttle compen-sates for the decreased glycine decarboxy-lation by dissipating the excess reducingequivalents (Gardeström et al., 2001).
In cotton leaves, maintained at low O2
concentration, a nonphotorespiratory con-dition, photosynthesis, was severely inhib-ited under strong light, compared with theones kept at normal O2 levels. The low O2samples also had reduced levels of chloro-plastic Pi. When Pi was fed to these leaves,the rates of photosynthesis were restored tothe levels of those kept in normal air con-taining 21% O2. This indicates that Pi limi-tation could partly be alleviated byphotorespiratory recycling of Pi. Thus, pho-torespiration reduces photoinhibition bykeeping up rates of photosynthesis throughmaking Pi available for the process (Guo etal., 1995).
Mitochondria, being major players inthe photorespiratory pathway, have to inter-act with chloroplasts and peroxisomes andtake part in balancing the photosyntheticredox equivalents and protection againstphotoinhibition.
D. Significance UnderTemperature or Water Stress
In addition to protection from photo-inhibition, mitochondria also help to opti-mize photosynthesis under stress conditions
like chilling temperature or osmotic stress(Table 5). After a period of cold hardening,the leaves of winter rye exhibited an in-crease in the rates of dark respiration inlight along with those of photosynthesis(Hurry et al., 1995). Oligomycin treatmentresulted in the inhibition of photosynthesismore in cold hardened leaves than that innonhardened ones, suggesting that the in-crease in photosynthetic capacity followingcold hardening is contributed to by mito-chondria. A similar situation of increasedtolerance to photoinhibition following coldhardening has been reported in the leaves ofwinter and spring wheat (Hurry and Huner,1992).
Circumstantial evidence points out tothe possible roles of AOX during the main-tenance of photosynthesis in low tempera-ture. The level of alternative oxidase pro-tein in tobacco (Vanlerberghe and McIntosh,1992) as well as the capacity of alternativerespiration (Rychter et al., 1988) usuallyincrease at low temperatures. The extent ofelectron partitioning to the alternative oxi-dase raises significantly at low tempera-tures in cold grown mung bean (Gonzàlez-Meler et al., 1999). These results indicate arole for alternative respiratory pathway inprotecting the plant tissues from chillingand related photoinhibition and suggest ageneral increase in alternative respirationunder stress conditions.
However, Ribas-Carbo et al. (2000) havefound increased electron flow in the alter-native pathway following cold treatment ina chilling sensitive cultivar of maize com-pared with a chilling tolerant one, indicat-ing no specific role for the alternative path-way of respiration in conferring chillingtolerance. Similarly, no specific increase inalternative respiration ocurred followingchilling in soybean cotyledons (Gonzàlez-Meler et al., 1999). Further studies and di-rect evidence are needed to assign any di-rect role of alternative pathway in the
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protection of photosynthesis from chillingstress in plants.
Mitochondrial respiration seems to berelated to decreased photosynthesis and in-creased susceptability to photoinhibitionunder osmotic stress. Mesophyll protoplastsof pea kept in hyperosmotic medium werehighly susceptable to photoinhibition whenthey were exposed to photoinhibitory light.On exposure to hyperosmotic medium at0°C, both photosynthetic and respiratoryrates decreased, indicating a correlationbetween the two processes (Saradadevi andRaghavendra, 1994). However, at 25°C, res-piration increased, while photosynthesis de-
creased. More experiments are needed tounderstand the role of respiration vis-a-visphotosynthesis during osmotic stress undervarying temperatures.
IV. OPTIMIZATION OFPHOTOSYNTHETIC CARBONASSIMILATION
The optimization of photosynthetic car-bon assimilation requires a coordination ofdifferent components: generation and use ofassimilatory power (ATP and NADPH),
TABLE 5Correlation Between the Pattern of Changes in Respiration and the Extent ofPhotoinhibition in Different Plant Tissues in Presence of Mitochondrial Inhibitors orStress Conditions. Any Decrease in Respiratory Activity Leads to an Increase in theExtent of Photoinhibition, While an Increase in Respiration Helps to Decrease thePhotoinhibition
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induction of photosynthesis, activation ofenzymes, and maintenance of metabolitelevels. In a photosynthesizing cell the mito-chondrial respiratory system may benefitdifferent components of chloroplast photo-synthesis by modulating any of the abovecomponents. However, emphasis has alreadybeen made on the significant role of mito-chondria in maintaining either cytosolic re-dox status or ATP or both (Krömer et al.,1988; Krömer and Heldt, 1991a; Krömer etal., 1993; Raghavendra et al., 1994;Gardeström and Lernmark, 1995; Krömer,1995; Igamberdiev et al., 1998; Padmasreeand Raghavendra, 1998).
A. Sustenance of SucroseBiosynthesis: Role of ATP
The biosynthesis of sucrose, one of themajor end products of photosynthetic car-bon assimilation, occurs in the cytosol ofmesophyll cells. Sucrose biosynthesis in thecytosol requires a continuous supply of car-bon skeletons and energy. Although it isobvious that chloroplasts play a significantrole in supplying the carbon compounds forthe synthesis of sucrose, the relative impor-tance of mitochondria in meeting the cyto-solic demands for ATP, particularly duringsucrose formation is emphasized only dur-ing the last decade (Raghavendra et al., 1994;Gardeström and Lernmark, 1995; Krömer,1995; Hoefnagel et al., 1998; Padmasreeand Raghavendra, 1998; Atkin et al., 2000b).Studies with a starchless mutant NS 458 ofNicotiana tabacum (defective in plastidphosphoglucomutase) in the presence of oli-gomycin also suggested that the mitochon-drial supply of ATP could affect assimilatepartitioning into sucrose and thereby modu-late photosynthesis (Hanson, 1992).
The transfer of redox equivalents gener-ated during the oxidation of TCA cycle in-
termediates along the mitochondrial elec-tron transport chain accumulate significantamounts of ATP in the mitochondrial ma-trix. Mitochondria have a very high capac-ity for ATP synthesis, in fact higher thanthat of chloroplasts, producing up to 3 ATPper NAD(P)H compared with 1.5 to 2.0ATP per NAD(P)H in the chloroplast(Hoefnagel et al., 1998; Siedow and Day,2000). It is possible that the ATP poolsgenerated in the mitochondrial matrix aretranslocated to cytosol (through adenylatetranslocator) to be used in sucrose synthesisor even imported into chloroplasts to beused in various other biosynthetic processeslike protein synthesis, NH4+ assimilation,metabolite transport, and maintenance(Hoefnagel et al., 1998).
It is possible in light that mitochondrialrespiration is subjected to adenylate control(Hoefnagel et al., 1998). However, the abil-ity of plant mitochondria to switch betweenthe rotenone-sensitive and rotenone-insen-sitive as well as the cyanide-sensitive cyto-chrome pathway and cyanide-resistant al-ternative pathways provides for a flexiblesystem and ATP production. However, thedegree to which mitochondrial ATP supplyin the light required for optimal photosyn-thesis depends on the balance of ATP pro-duction and consumption in chloroplasts.Two key observations indicate the primaryrole of mitochondria in assisting chloro-plasts in meeting the cytosolic demands ofATP for sucrose synthesis: (1) An increasein cytosolic or cellular levels of glucose-6-P and other phosphates (e.g., fructose-6-Pand fructose-2,6-bisphosphate) in the pres-ence of oligomycin or antimycin A (Krömerand Heldt, 1991a; Krömer et al., 1993,Padmasree and Raghavendra, 1999c). Sub-cellular analysis of protoplasts revealed thatthe increase in hexose monophosphates wasmostly in the cytosol, demonstrating therestriction of sucrose biosynthesis (Krömeret al., 1992); (2) Restriction of mitochon-
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drial ATP synthesis by oligomycin or an-timycin A or photorespiratory glycine oxida-tion using AAN in isolated protoplasts causeda marked reduction in ATP/ADP ratios in thecytosolic and mitochondrial compartmentsthan that of chloroplasts (Gardeström et al.,1981; Gardeström and Wigge, 1988; Krömerand Heldt, 1991a; Krömer et al., 1993;Igamberdiev et al., 1998).
The change in the levels of intracellularATP and ADP during illumination causedby mitochondrial inhibitors at limiting CO2was in contrast to that of photosynthesis.Despite the expectation that ATP demandswould be low at limiting CO2, there was asteep positive correlation between the ratesof photosynthesis and ratios of ATP to ADPin protoplasts in the presence of oligomycinor antimycin A but not SHAM (Figure 7).The Glc-6-P level increased by about 19 to30% in the presence of both oligomycin andantimycin A at optimal CO2 conditions. The
marked increase in Glc-6-P in mesophyllprotoplasts in the presence of only oligomy-cin or antimycin A but not SHAM suggeststhat the cytochrome pathway of electrontransport (and oxidative phosphorylation)modulates sucrose biosynthesis, while thealternative pathway may not have a signifi-cant role (Padmasree and Raghavendra,1999a).
The restriction of mitochondrial ATPsynthesis by oligomycin and antimycin Awould not only limit sucrose synthesis butalso cause feedback inhibition of photosyn-thetic activity because the phosphatetranslocator in the inner chloroplast mem-brane is regulated by the equilibrium of thetriose-P concentration in the stroma and thecytosol. However, an elevated cytosolic levelof DHAP or reduced flux of DHAP fromchloroplast can also lead to decreased stro-mal PGA level and thereby decreased Calvincycle activity (Krömer et al., 1993,
FIGURE 7. Positive correlation occurs between the ratios of ATP/ADP and the relative rates ofphotosynthesis in pea mesophyll protoplasts in presence of antimycin A (inhibitor of cytochromepathway in mitochondria) but not SHAM, (inhibitor of alternative pathway). In other words, SHAMwhich markedly inhibits photosynthesis of protoplasts, does not alter the relative ratio of ATP/ADP.(Adapted from Padmasree and Raghavendra, 1999a.)
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Padmasree and Raghavendra, 1999a; Flüggeand Heldt, 1991). Thus, mitochondrial oxi-dative electron transport plays a significantrole in optimizing photosynthetic carbonassimilation by sustaining sucrose biosyn-thesis. The flexibility of mitochondrial elec-tron transport chain to meet cytosolic de-mands under both dark and light conditionsmakes it a ready source of energy to meetcellular needs supplementing the chloroplastmetabolism.
B. Maintenance of CellularRedox State: Ratios of Malate/OAA and Triose-P/PGA
Chloroplasts always have a tendency toget overreduced as the rate of photochemi-cal reaction and utilization of reducing po-tential in metabolism have been estimatedto differ by at least 15 orders of magnitude(Huner et al., 1998). It is essential that theexcess reduced equivalents are taken out ordisspiated quickly to prevent damage to thethylakoid membranes (Gillmore, 1997;Niyogi et al., 1998; Niyogi, 1999).
Mitochondria also appear to play a sig-nificant role in maintaining optimal levels ofredox equivalents in the chloroplasts to keepup the Calvin cycle activity, possibly by co-ordinating with peroxisomes and cytosol. Thereductants in excess of the requirements ofthe Calvin cycle are exported out of chloro-plasts through the shuttling of either OAA-malate (by dicarboxylate translocator) orPGA-DHAP (Pi-translocator).
The relative levels of triose-P/PGA andmalate/OAA reflect the redox state of cyto-sol and the cell. Mitochondrial electron trans-port appears to be one of the efficient pro-cesses to use up the reduced equivalents.Any limitation on the mitochondrial me-tabolism leads to a marked rise in the redoxstate of cells, as indicated by the rise in the
ratios of malate/OAA or triose-P/PGA(Padmasree and Raghavendra, 1999c).
The steep gradient in redox levels be-tween stromal compartment and cytosol ismaintained by regulation at several steps suchas (1) chloroplastic NADP-MDH, (2) triose-P/Pi translocator, (3) glycolate/glyceratetranslocator, and (4) glycine/serine translocator(Gardeström et al., 2001). Among these,NADP-MDH functions like a valve, releasingthe excess reductant from chloroplasts asmalate (Scheibe, 1991). Malate valve allowschloroplasts also to provide reducing equiva-lents either to peroxisomes for reduction ofhydroxypyruvate (under photorespiratory con-ditions, Krömer, 1995) or mitochondria to beoxidized by the internal NADH-dehydroge-nase system (under nonphotorespiratory con-ditions; Padmasree and Raghavendra, 1998).This would still allow some of the NADHformed during glycine decarboxylation to beretained in the mitochondria, rather than shut-tling it to the peroxisome to supporthydroxypyruvate reduction. As a result, NADHcan be oxidized within the mitochondria toprovide additional ATP for extrachloroplasticprocesses, such as sucrose synthesis or reduc-tion of PGA in the cytosol (Krömer and Heldt,1991a,b).
The operation of cyanide-resistant alter-native and cyanide-sensitive cytochromepathways of mitochondria appear to be closelyintegrated with the redox regulation duringphotosynthetic metabolism (Padmasree andRaghavendra, 1999c). Restriction of a cya-nide-resistant pathway by SHAM markedlyelevated the malate/OAA ratios, while therestriction of cyanide-sensitive pathway byantimycin A or oligomycin lead to a markedincrease in triose-P/PGA ratios (Figure 8).Because an accumulation of malate repre-sents an overreduction of chloroplasts(Backhausen et al., 1994), the marked in-crease in malate/OAA in the presence ofSHAM suggests an accumulation of redoxpower in protoplasts when AOX pathway is
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FIGURE 8. Changes in the redox state of pea mesophyll protoplasts during photosynthesis in theabsence or presence of typical inhibitors of mitochondrial electron transport. On illumination inpresence of 1.0 mM bicarbonate, the ratios of Triose-P/PGA or Malate/OAA rise with time indicatingthe increase in the redox state of protoplasts. The presence of 250 nM antimycin A (inhibitor ofcytochrome pathway) results in the preferential accumulation of triose-P, while the presence of 200µM SHAM (inhibitor of alternative pathway of mitochondrial electron transport) causes the accumu-lation of malate. (Adapted from Padmasree and Raghavendra, 1999c.)
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restricted. Thus, AOX appears to promotethe consumption of malate in pea mesophyllprotoplasts.
C. Shortening of Induction
The phenomenon of induction (delay inachieving maximal rates) is a common fea-ture of photosynthesis (Edwards and Walker,1983; Walker, 1988). Among the most im-portant factors that cause photosynthetic in-duction are the activation of key chloroplas-tic enzymes (including NADP-malatedehydrogenase, NADP-glyceraldehyde3-phosphate dehydrogenase, stromalFBPase, PRK) and the autocatalytic build-up of Calvin cycle metabolites, for example,
RuBP (Salvucci, 1989; Scheibe, 1991;Edwards and Walker, 1983).
Mitochondrial contribution to photo-synthetic metabolism during photosyn-thetic induction was investigated in me-sophyll protoplasts from barley or pealeaves by using rotenone or oligomycin(Table 6). Both the inhibitors increasedthe lag phase of photosynthetic inductionduring the transition of protoplasts fromdarkness to light (Igamberdiev et al.,1998). Prolongation of photosynthetic in-duction period was observed also withantimycin A, SHAM, and propyl gallate(Figure 9). However, SHAM and propylgallate (inhibitors of alternative pathway)had a negligible effect on the photosyn-thetic induction period (Padmasree andRaghavendra, 1999b).
TABLE 6Prolongation of Photosynthetic Induction in Mesophyll Protoplasts as a Consequence ofRestriction of Mitochondrial Metabolism. The Lag Period for Reaching the Maximum Rateof Photosynthetic Carbon Assimilation (in Presence of 1.0 m M Bicarbonate) is Extendedby the Presence of Mitochondrial Inhibitors
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FIG
UR
E 9
. Pro
long
atio
n of
pho
tosy
nthe
tic in
duct
ion
in p
rese
nce
of ty
pica
l inh
ibito
rs o
f mito
chon
dria
l tra
nspo
rt. T
he la
gpe
riod
for
reac
hing
the
max
imum
rat
e of
pho
tosy
nthe
sis
afte
r sw
itchi
ng o
n th
e lig
ht (
indi
cate
d by
‘L’)
is u
sual
ly le
ss th
an3
min
, w
hile
thi
s la
g in
crea
ses
to a
lmos
t 5
or 8
min
in p
rese
nce
of o
ligom
ycin
(1
µg m
l–1)
or a
ntim
ycin
A (
1 µM
). T
heex
act r
easo
ns fo
r su
ch m
arke
d in
crea
se in
the
phot
osyn
thet
ic in
duct
ion
perio
d ar
e no
t cle
ar. I
t cou
ld b
e al
so o
ne o
f the
cons
eque
nces
of
depr
essi
on i
n ca
rbon
ass
imila
tion
by m
itoch
ondr
ial
inhi
bito
rs.
(Ada
pted
fro
m P
adm
asre
e an
dR
agha
vend
ra,
1999
b.)
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Despite the apparent surplus of ATP inthe chloroplast, the demands for ATP dur-ing the initial phase of photosynthetic in-duction are met by mitochondrial oxidativephosphorylation. The delay by rotenone andoligomycin in photosynthetic induction ap-pears to be caused by a restriction on thereoxidation of redox equivalents from thechloroplasts (and associated reactions) bythe mitochondrial electron transport chain(Igamberdiev et al., 1998). This hypothesismay indeed be complemented by the negli-gible changes in the RuBP levels associatedwith prolonged induction period in the pres-ence of oligomycin and antimycin A in pealeaves (Padmasree and Raghavendra,1999b). The marked sensitivity of photo-synthetic induction period to rotenone orantimycin A suggests that the redox equiva-lents from chloroplasts are being oxidizedby internal dehydrogenase via complex Iand complex III, but not through the roten-one-insensitive dehydrogenases. One of thebenefits of the mitochondrial oxidative elec-tron transport coupled to oxidativephosphorylation is the maintenance or mini-mization of the induction phase of photo-synthesis. The negligible effect on photosyn-thetic induction by SHAM or propyl gallatecompared with the marked delay by antimy-cin A or oligomycin suggests that electrontransport via alternative oxidase may not beas significant as that of cytochrome pathwayduring photosynthetic induction.
Thus, the restriction of mitochondrialactivity leads to an increase in the photo-synthetic induction period in mesophyllprotoplasts of pea as well as barley(Igamberdiev et al., 1998; Padmasree andRaghavendra, 1999b). However, this cor-relation may be incidental because thereis no direct evidence to suggest that re-stricted mitochondrial metabolism is thecause for the prolongation of photosyn-thetic induction.
D. Activation of Enzymes
When leaves are illuminated, the acti-vation state/activity of not only chloroplas-tic enzymes but of several enzymes locatedin different compartments of the cell isstimulated. In this review, attention is drawnto the enzymes involved in coordinating theinteractions between chloroplasts, mitochon-dria, peroxisomes, and cytosol. These en-zymes located in different compartments ofthe cell are fine tuned and coordinated witheach other by specific metabolites.
The light activation of photosyntheticenzymes located in stroma is regulated byseveral factors: ferredoxin-thioredoxin sys-tem, metabolite levels, pH, ionic status, andoxidation-reduction potential (Scheibe, 1990;Faske et al., 1995). Rotenone and oligomy-cin both delayed the activation of chloroplas-tic NADP-MDH during the transition fromdarkness to light (Igamberdiev et al., 1998).The timing of the delay in activation ofNADP-MDH is very similar to the delay inphotosynthetic O2 production and the delayin build-up of nonphotochemical quenching.Restriction of mitochondrial electron trans-port delays alkalization of the chloroplaststroma, which in turn delays the activation ofNADP-MDH and there by the export (andthus use) of redox equivalents (Igamberdievet al., 1998). A marked decrease in lightactivation of not only NADP-MDH, but alsoFBPase, NADP-GAPDH, and PRK in pres-ence of SHAM during steady state photosyn-thesis indicates that the alternative pathwaymay play a significant role in maintaining theactivation status of chloroplastic enzymes(Padmasree and Raghavendra, 2001a). Thisis possible by regulating the redox equiva-lents through malate-OAA shuttle.
Apart from chloroplastic enzymes, cy-tosolic enzymes can also be regulated bylight. One of such is sucrose phosphate syn-
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thase (SPS), which plays a significant roleis sucrose biosynthesis. SPS is subjected toreversible phosphorylation-dephosphoryla-tion cascade. The dephosphorylated enzymeis more active than the phosphorylated from(Huber and Huber, 1996). At high CO2, thedecrease in the mitochondrial and cytosolicATP/ADP ratios caused by the oligomycintreatment at high and low irradiance canlead to a decrease in SPS activity (Krömeret al., 1993). Under high CO2, this inhibi-tion of sucrose synthesis by oligomycin ap-parently increased cytosolic Glc-6-P levelsand caused feedback inhibition of the Calvincycle and photosynthetic activity.
E. Integration withPhotorespiration and NitrogenMetabolism
The interaction of mitochondrial respi-ration, nitrogen metabolism, and photosyn-thesis has been the subject of two extensivereviews (Padmasree and Raghavendra, 1998;Gardestrom et al., 2001). The interplay ofthese three metabolic pathways involvesrecycling of carbon, nitrogen, and markedamounts of reduced equivalents and someATP. The rapidity in the turnover (produc-tion and consumption) of NAD[P]H, reducedferredoxin and ATP allows them to coordi-nate at least four different metabolic path-ways viz., photosynthesis, respiration, pho-torespiration, and nitrogen metabolism.Apart from CO2 fixation, the second largestsink for photosynthetic energy in manyhigher plants is nitrate. As the assimilationof nitrate in many species occurs predomi-nantly in the leaves, this process will oftenbe ongoing simultaneously with CO2 fixa-tion in photosynthetic cells (Noctor andFoyer, 1998).
Three processes related to photorespira-tion and nitrogen metabolism form impor-
tant links between chloroplasts, mitochon-dria, and peroxisomes. These are (1) gly-cine oxidation; (2) reductive amination ofoxoglutarate, and (3) hydroxypyruvate re-duction. These three processes are highlycoupled, and modulation of any one of themleads to a cascading effect on the other.
Glycine is formed in peroxisomes andoxidized in mitochondria. The precursor ofglycine is glycolate from chloroplasts. Gly-cine oxidation yields considerable amountsof NADH and NH4, besides CO2. The result-ing NADH is either used up for ATP produc-tion or exported out in the form of malate tomeet the requirements of hydroxypyruvatereduction in peroxisomes (Heldt et al., 1998;Raghavendra et al., 1998).
Oxidation of photorespiratory glycine iscoupled to hydroxypyruvate reduction(Hanning and Heldt, 1993; Heldt et al., 1998;Raghavendra et al., 1998). Any uncoupling ofglycine oxidation and hydroxypyruvate reduc-tion would imply a huge photorespiratory pro-duction of ATP, particularly in the mitochon-dria. If metabolic conditions demand, the extraNADH is used also for nitrate reduction incytosol. In algae, Weger et al. (1988) haveshown that high rates of NH4
+ assimilation areassociated with a marked increase in cyanide-sensitive O2 uptake.
The NADH generated by the glycinedecarboxylase reaction is expected to ac-count only for 50% of the reducing powerused in a subsequent reduction of thephotorespiratory hydroxypyruvate in per-oxisomes (Krömer and Heldt, 1991b). Theremainder of the reducing power is pro-vided by photosynthetic processes in thechloroplast (Krömer and Heldt, 1991b;Igamberdiev and Kleczkowski, 1997). Gly-cine oxidation can also increase theintramitochondrial and cytosolic ATP/ADPratio (Gardeström and Wigge, 1988); there-fore, the mitochondrial respiratory chain canplay a role in the cellular ATP production inthe light. Glycine oxidation in mitochondria
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of photosynthetic tissue in leaves of wheatand maize are coupled in more degree withcyanide-resistant and rotenone-resistantpaths of electron transport contrary to eti-olated leaves, where these pathways are in-volved to a much less extent (Igamberdievand Kleczkowski, 1997c; Igamberdiev etal., 1998).
Besides CO2, the other major sinks forreducing equivalents in chloroplasts are themetabolic reactions involving the reductionof nitrogen or sulfur. The reduction ofoxoglutarate to glutamate (or nitrite to NH4
+)occurs exclusively in chloroplasts. The majorsubstrate for these reactions, namely,oxoglutarate is exported from mitochondria,which operate a partial TCA cycle in light(see Section I.D). A continuous supply ofoxoglutarate from mitochondria is requiredfor NH4 assimilation into amino acids inchloroplasts. Similarly, the supply of nitriteto chloroplasts is also dependent on mito-chondrial activity, which provides signifi-cant amounts of NADH for nitrate reduc-tion in cytoplasm (Weger and Turpin, 1989;Padmasree and Raghavendra, 1998). Therequired NADH in mitochondria is gener-ated from glycine coming from peroxisomes.Thus, chloroplasts, mitochondria, and per-oxisomes have to work together to keep upthe reduction of nitrite and reductiveamination of oxoglutarate (Figure 10).
Glycine and malate, both of which areformed during active photosynthesis, form thesubstrates for leaf mitochondrial oxidation invivo. However, the main substrate for mito-chondrial respiration in the light is probablyglycine, which is produced at high rates duringphotorespiration. At least 25% of the NADHformed during oxidation of these metabolites isused for extra-mitochondrial requirements, par-ticularly hydroxypyruvate reduction in peroxi-somes and NO3– reduction in cytosol. The ex-port of reducing equivalents from mitochondriamay proceed by either a malate-aspartate shuttleor a malate-OAA shuttle. Chloroplasts form
alternative sources of reducing equivalents.Cytosolic nitrate reductase (NR) and peroxiso-mal hydroxypyruvate reductase can be servedvia a chloroplastic malate-OAA shuttle withreducing equivalents generated from photosyn-thetic electron transport (Heupel and Heldt,1992). Thus, mitochondrial metabolism be-comes a very important link among photosyn-thesis, photorespiration, and nitrogen assimila-tion in recycling NH4
+, reduced equivalents,and carbon skeletons (Figure 10).
F. Role in C 4 photosynthesis
Mitochondria play a direct role in car-bon metabolism of certain C4 and CAMplants, particularly those utilizing NAD-malic enzyme or PEP carboxykinase for C4-acid decarboxylation. In these plants thedecarboxylation of malate or aspartate oc-curs in mitochondria. During this function,mitochondria supply not only the carbonskeletons but also extra ATP needed for C4
pathway. The photosynthetic rates attainedby NAD+-malic enzyme plants suggest thatcarbon flux through the bundle sheath mito-chondria is 10- to 20-fold greater than thestandard respiratory carbon flux, andseveralfold greater than the flux of glycinethrough the mitochondria of C3 plants dur-ing photorespiration. Further, the NADHgenerated by NAD+-malic enzyme is uti-lized also for ATP synthesis. The predictedstoichiometry is about two malate moleculesoxidized per five molecules of PEP pro-duced (Siedow and Day, 2000). In PEPcarboxykinase type plants, the situation ismore complex.
The C4 plants do not show any photo-respiration because the process is confinedto bundle sheath cells and any CO2 releasedout is refixed efficiently in the surroundinglayer of mesophyll cells. Thus in C4 plants,mitochondrial location of GDC and result-
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FIG
UR
E 1
0. T
he i
nter
rela
tion
betw
een
nitr
ogen
met
abol
ism
, ch
loro
plas
t ph
otos
ynth
etic
rea
ctio
ns,
and
the
mito
chon
dria
lre
spira
tory
act
ivity
in p
lant
cel
ls. T
he in
itial
ste
p of
nitr
ate
redu
ctio
n to
nitr
ite o
ccur
s in
cyt
opla
sm. T
he m
ajor
ste
ps o
f for
mat
ion
and
assi
mila
tion
of a
mm
onia
are
loca
ted
in c
hlor
opla
sts.
The
red
ucin
g po
wer
for
nitr
ogen
ass
imila
tion
is s
uppl
ied
from
bot
hch
loro
plas
ts a
nd c
ytos
ol. T
he p
rovi
sion
of c
arbo
n sk
elet
ons
for a
mm
onia
ass
imila
tion
as w
ell a
s th
e re
cycl
ing
of p
hoto
resp
irato
ryam
mon
ia is
faci
litat
ed b
y m
itoch
ondr
ia. T
he k
ey e
nzym
es in
volv
ed in
thes
e pr
oces
ses
are
CS
, Citr
ate
synt
hase
; GD
C, G
lyci
nede
carb
oxyl
ase;
GO
GA
T,
Glu
tam
ate
oxog
luta
rate
am
ino
tran
sfer
ase/
Glu
tam
ate
synt
hase
; G
P,
Pho
spho
glyc
eral
dehy
de d
ehy-
drog
enas
e; G
S, G
luta
min
e sy
nthe
tase
; ID
H, I
soci
trat
e de
hydr
ogen
ase;
MD
H, M
alat
e de
hydr
ogen
ase;
ME
, Mal
ic e
nzym
e; N
iR,
Nitr
ite r
educ
tase
; N
R,
Nitr
ate
redu
ctas
e; P
DC
, P
yruv
ate
deca
rbox
ylas
e; P
EP
C,
Pho
spho
enol
pyru
vate
car
boxy
lase
; P
K,
Pyr
uvat
e ki
nase
.
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ing CO2 efflux becomes a crucial factor.Only the mitochondria located in bundlesheath cells of C4 palnts possess GDC, butnot those of mesophyll cells. The inter- andintracellular localization of GDC thus fa-cilitates the function of not only C4 photo-synthesis but also C3-C4 intermediacy (Deviet al., 1995; Rawsthorne, 1998).
V. BIOCHEMICAL BASIS:INTERORGANELLEINTERACTION
Rapid movement of metabolites occursbetween chloroplasts, cytoplasm, mitochondria,and peroxisomes. Such metabolite movementforms an important basis of interorganelle in-teraction as well as the optimization of differentmetabolic pathways in a plant cell. Severalinvestigators therefore attempted to study themetabolite patterns as the biochemical basis ofessentiality of mitochondrial respiration for pho-tosynthetic carbon assimilation, under variedconditions, for example, limiting or saturatingCO2, variable light intenstity (Krömer et al.,1988; Krömer et al., 1992; Krömer et al., 1993;Igamberdiev et al., 1997a,b, 1998; Padmasreeand Raghavendra, 1999a,b,c). These metabo-lites can be categorized into four groups:
1. Metabolites related to redox status, e.g.,malate or triose-P (mainly DHAP)
2. Adenylate compounds such as ATP orADP
3. Metabolites related to sucrose biosyn-thesis
4. Intermediates of the Calvin cycle
A. Major Products of OrganelleMetabolism
The interaction between the chloroplastsand mitochondria often involves not only
cytosol but also peroxisomes. Within thecell, there is always a high demand for ATPand reducing equivalents. The responsibil-ity of meeting the cellular requirements ofATP and NADH is shared by both chloro-plasts and mitochondria. The metabolismwithin chloroplasts, mitochodria, or peroxi-somes is optimized only when these or-ganelles are able to export their metabolitesand keep up the interorganelle metabolitemovement. The export of reduced equiva-lents from chloroplasts is essential to pre-vent overreduction of chloroplasts.
1. Chloroplasts
The major products exported from chlo-roplasts of C3 plants in light are glycolate,triose-P, and malate. The pattern of exportdepends on the carboxylase vs. oxygenaseactivity of Rubisco, which in turn dependson the ambient CO2. While the carboxylaseactivity of Rubisco results in the formationof triose-P, the oxygenase activity of Rubiscoleads to the formation of glycolate. Becausethe ratio of oxygenation to carboxylationduring photosynthesis in a leaf is 0.2 to 0.5,very high metabolic flux of glycolate oc-curs through the leaf peroxisomes (Heupelet al., 1991; Reumann et al., 1994). About50 to 75% of carbon from glycolate is sal-vaged in a sequence of photorespiratoryreactions that involves cooperation betweenthe chloroplasts, the mitochondria, and theperoxisomes.
Part of the triose-P formed by the reduc-tion of 3-PGA in chloroplasts is used up toregenerate RuBP, while the majority is ex-ported out to be utilized for either sucrosesynthesis or respiratory glycolytic pathway.Triose-phosphates are exported in exchangefor Pi through triose-P-Pi translocator lo-cated on the chloroplast inner membrane(Flügge, 1999). Triose-P exported mostly inthe form of DHAP to cytosol serves two
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major purposes: (1) form sucrose, (2) oxi-dation to PGA releasing ATP and NADH tomeet the needs of cytosol.
In light, the export of malate from chloro-plasts plays a significant role in the transfer ofreducing equivalents formed in excess of thoserequired to operate Calvin cycle. When theNADPH/NADP+ ratio in the chloroplast ishigh, OAA is converted to malate and ex-ported via the dicarboxylate translocator inthe inner envelope membrane of the chloro-plasts (Heineke et al., 1991). For the malate/OAA shuttle to operate as an effective NADPHexport system, the exported malate must beoxidized to regenerate OAA for transport backto the chloroplast. Thus, malate released intothe cytosol is either oxidized in cytosol tosupport nitrate reduction or transferred to per-oxisomes to support hydyroxypyruvate reduc-tion (Atkin et al., 2000b). Under conditionswhere more reductant is produced than is re-quired for cytosolic and peroxisomal processes,malate can be imported into mitochodria foroxidation and allow ATP synthesis (Hoefnagelet al., 1998).
Among the products of chloroplast me-tabolism, glycolate is the substrate forphotorespiratory metabolism, which helpsin the dissipation of excess energy as wellas protection against photoinhibition (seeSection III.C). Triose-P and malate facili-tate export of the reducing power and ATPfrom chloroplasts and thus act as sinks. Atlimiting CO2 malate is the major carrier ofreducing equivalents sent out of chloroplasts,while at optimal CO2, triose-P becomes thedominant carrier of reducing equivalents andleads to the formation of sucrose.
2. Peroxisomes
Glycine and glycerate are the majorproducts exported from peroxisomes. In theperoxisomes, glycolate is oxidized to
glyoxylate and then to glycine usingglutamate as the amino donor.
Glycine is exported from peroxisomesto mitochondria. On the other hand, peroxi-somes import serine from mitochondria andconvert it to hydroxypyruvate. The reduc-tion of hydroxypyruvate leads to the forma-tion of glycerate, which is exported to chlo-roplasts, facilitating the salvage of carbon.The reduction of hydroxypyruvate toglycerate places a high demand for reduc-ing equivalents. This demand is met in theform of malate exported to peroxisomes fromboth chloroplasts and mitochondria (Heldtet al., 1998; Raghavendra et al., 1998).
The metabolites within the peroxisomesare channelled through multienzyme com-plexes located in the matrix of peroxisomes.The metabolite movement into and out ofperoxisomes occurs through specific porescalled ‘porins’ (Reumann et al., 1995).
3. Mitochondria
The three major compounds exportedfrom mitochondria are serine (participatesin photorespiratory cycle), oxoglutarate (tosupply carbon compounds for N2 metabo-lism), and malate (to transfer reducingequivalents to peroxisomes).
The glycine formed in the peroxisomesis transported into the mitochondria, whereit is oxidized by glycine decarboxylase-serine hydroxymethyl transferase complexto yield serine, CO2, NH+
4, and NADH(Oliver, 1998; Douce and Neuberger, 1999).Serine leaves the mitochondria via a spe-cific translocator, possibly the sametranslocator by which glycine is taken up.
Carbon intermediates, particularly2-oxoglutarate, are exported from the TCAcycle to support GOGAT activity forglutamate synthesis in the chloroplasts (Fig-ure 10). While the major route of
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2-oxoglutarate production involves partialoperation of the TCA cycle in the mito-chondrion, 2-oxoglutarate synthesis mayalso occur via a cytosolic isocitrate dehy-drogenase (see Section I.D., Figure 2). Inmature leaves, the most important input ofC4 acids for 2-oxoglutarate synthesis ap-pears to be as oxaloacetate, generated bycytosolic phosphoenolpyruvate carboxylase.Approximately half of the PEP available incytosol is carboxylated by PEPC to oxalo-acetate, which is converted to 2-oxoglutaratethrough reactions catalyzed by citrate syn-thase, aconitase and isocitrate dehydroge-nase (Foyer et al., 2000).
In contrast to mitochondria from animaltissues, whose inner membrane is impermeableto oxaloacetate, the plant mitochondrial innerenvelope membrane has a malate-oxaloacetatetranslocator that facilitates the exchange ofmalate and oxaloacetate (Ebbighausen et al.,1985; Douce and Neuburger, 1990). The highactivity of malate dehydrogenase in the mito-chondrial matrix ensures an efficient reductionof oxaloacetate to malate. Thus, the NADHformed during glycine oxidation is incorpo-rated into malate and is exported by the malate-oxaloacetate shuttle. This shuttle has a highcapacity to transfer reducing equivalents frommitochondria. Although the amount of NADHgenerated in the mitochondria from glycineoxidation is quite high, mitochondria deliveronly about half the reducing equivalents re-quired for peroxisomal hydroxypyruvate re-duction, while the rest is provided by the chlo-roplasts (Heldt et al., 1998; Padmasree andRaghavendra, 2000).
B. Metabolite Exchange betweenChloroplasts, Mitochondria,Peroxisomes, and Cytosol
ATP and NAD(P)H are required in sev-eral steps of metabolic reaction occurring in
different cellular compartments. However,ATP and NAD(P)H being not permeableacross the membrane have to be transportedindirectly through different metaboliteshuttles. The rapid exchange of metabolitesbetween chloroplasts, mitochondria, peroxi-somes, and cytosol according to the cellularneeds of energy demand is the biochemicalbasis as well as essential component ofinterorganelle interaction (Figure 11).
During illumination, the difference inredox potentials between the stromal com-partment (NADPH/NADP) and cytosol(NADH/NAD) is quite large, leading to thetransfer of redox equivalents from the chlo-roplast stroma to the cytosol (Heineke et al.,1991). The transfer of reducing equivalentsfrom chloroplasts is mediated by two differ-ent metabolite shuttles: the triose-P-PGAshuttle mediated by the phosphate translocatorand the malate-OAA shuttle facilitated bythe dicarboxylate translocator. The triose-P-PGA shuttle is regulated by Pi availabilityfor counter-exchange by the phosphatetranslocator, as well as PGA reduction inchloroplasts and triose-P oxidation in cyto-sol. On the other hand, the malate-OAAshuttle is regulated by stromal NADP-MDHand the [NADPH]/[NADP], and also by thetranslocating step across the innerchloroplastenvelope membrane. In addition, metaboliteshuttles of malate and OAA between mito-chondria and the cytosol as well as cytosoland peroxisomes facilitate further the ex-change of reducing equivalents between mi-tochondria, cytosol, and peroxisomes (Heldt,1997).
A photosynthetic cell has two differentsystems to produce and meet cytosolic de-mands of ATP: photophosphorylation andoxidative phosphorylation. The ATP pro-duced during photophosphorylation is trans-ferred from chloroplast to cytosol throughthe exchange of triose-P and PGA mediatedby triose-P-Pi translocator. An NAD-depen-dent glyceraldehyde phosphate dehydroge-
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FIG
UR
E 1
1. T
he b
ioch
emic
al b
asis
of i
nter
orga
nelle
inte
ract
ion
betw
een
chlo
ropl
asts
, cyt
osol
, mito
chon
dria
, and
per
oxis
omes
. The
rapi
d m
ovem
ent
of m
etab
olite
s be
twee
n th
ese
orga
nelle
s fa
cilit
ates
the
exp
ort
of r
educ
ed e
quiv
alen
ts a
s w
ell
as A
TP
fro
m c
hlor
opla
sts
and
mito
chon
dria
.P
erox
isom
es f
orm
a m
ajor
sin
k fo
r re
duce
d eq
uiva
lent
s, w
hile
AT
P is
nee
ded
for
seve
ral a
ctiv
ities
(in
clud
ing
the
sucr
ose
bios
ynth
esis
) in
cyt
osol
.T
he m
etab
olite
shu
ttle
is fa
cilit
ated
by
spec
ific
carr
ier
prot
eins
on
the
mem
bran
es, c
alle
d tr
ansl
ocat
ors,
whi
ch a
re in
dica
ted
by n
umbe
rs 1
to 6
. The
glyc
olat
e/gl
ycer
ate
tran
sloc
ator
(1)
loc
ated
on
inne
r ch
loro
plas
t m
embr
ane
and
glyc
ine/
serin
e tr
ansl
ocat
or (
2) l
ocat
ed o
n in
ner
mito
chon
dria
lm
embr
ane
coor
dina
te th
e m
etab
olite
traf
fic in
volv
ing
maj
or p
hoto
resp
irato
ry m
etab
olite
s, b
etw
een
chlo
ropl
asts
, per
oxis
omes
, and
mito
chon
dria
. The
othe
r tw
o m
ajor
gat
eway
s in
volv
ed a
re th
e P
i tra
nslo
cato
r (2
) an
d di
carb
oxyl
ate
tran
sloc
ator
(3)
in c
hlor
opla
sts.
In m
itoch
ondr
ia, t
he o
ther
cha
nnel
sar
e th
e ad
enyl
ate
tran
sloc
ator
(5)
and
oxa
loac
etat
e tr
ansl
ocat
or (
6). T
he m
itoch
ondr
ial e
lect
ron
tran
spor
t (E
TC
) sy
stem
can
oxi
dize
ext
erna
l as
wel
las
inte
rnal
NA
DH
and
gen
erat
e A
TP
. (A
dapt
ed f
rom
Pad
mas
ree
and
Rag
have
ndra
, 19
98.)
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nase plays a significant role in facilitatingavailability of the chloroplastic ATP to cy-tosolic demands (Krömer, 1995). On theother hand, ATP produced during oxidativephosphorylation in mitochondria can betransferred directly to cytosol through ade-nylate translocator.
Illuminated chloroplasts usually haveexcess NADPH or related metabolites be-cause their electron transport activity is inmuch excess of the capacity of carbon fixa-tion (Huner et al., 1998). The excess reduc-ing equivalents are transported from the chlo-roplasts (in the form of DHAP and malate)to the cytosol to generate NADH. Mito-chondria are capable of oxidizing externalNADH. However, the oxidation could alsobe indirect through the shuttles of relatedmetabolites formed during photosynthesis(Gardeström et al., 2001). For example, gly-colate/glycerate translocator of chloroplastsand glycine/serine translocator of mitochon-dria can channel large amounts of glycineinto mitochondria. As glycine is the preferedmitochondrial substrate over malate, underphotorespiratory conditions NADH gener-ated during glycine oxidation can besuccesfully oxidized through the nonphos-phorylating pathways of mitochondrial elec-tron transport even when ADP is limited.Half of the reducing equivalents producedin the mitochondrial matrix are transferredto peroxisomes in the form of malate tosupport hydroxypyruvate reduction, whilethe rest is supplemented by malate derivedfrom chloroplasts.
On the basis of the metabolite movementsdescribed above, the photosynthetic and res-piratory activity in chloroplasts and mitochon-dria, respectively, appears to be modulated byone or more of the following factors: (1) theredox state due to the relative levels of NAD(P)or NAD(P)H (b) interorganelle movement ofmetabolites such as PGA, DHAP, malate, andOAA and (c) adenine nucleotides (ATP, ADP).Peroxisomes and cytoplasm naturally and
closely linked to these processes and form anactive and integral components of metabolitemovements and subsequent interorganelle in-teraction.
VI. FUTURE PERSPECTIVES
Most of the experiments on the interac-tion between mitochondria and chloroplastsduring photosynthesis have been made withprotoplasts (e.g., Gardeström et al., 1992;Krömer et al., 1988, 1993; Igamberdiev etal., 1998; Padmasree and Raghavendra,1999a,b,c). Only a few experiments wereconducted with intact leaves (Krömer andHeldt, 1991a; Hanson, 1992; Hanning andHeldt, 1993; Hurry et al., 1995; Atkin et al.,1998). However, more experiments areneeded using intact tissues or leaf discs soas to understand and extrapolate the situa-tion in leaves because the isolated proto-plasts lack the cell wall typical of plant cellsand may deviate in their metabolism.
An inherent limitation of metabolic in-hibitors is the possibility of their unspecificand multiple effects on different processes inthe cell. For example, SHAM, used exten-sively to inhibit alternative oxidase pathway,may also affect chlororespiration (Singh etal., 1992), chloroplastic glycolate-quinoneoxidoreductase (Goyal and Tolbert, 1996),besides stimulating peroxidase (Lambers,1985; Møller et al., 1988). Similarly, antimy-cin A used to suppress cytochrome pathwaymay also affect chlororespiration (Singh etal., 1992) as well as photosynthetic O2 evolu-tion (Cornic et al., 2000) due to the interfer-ence with ferredoxin-dependent reduction ofcyt b-559 particularly in intact chloroplasts(Scheller, 1996; Endo et al., 1998; Ivanov etal., 1998) besides stimulation of carbon fixa-tion (Schacter and Bassham, 1972). There-fore, the use of inhibitors to examine the roleand importance of the cytochrome and alter-
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native pathways has been questioned fre-quently. Neverthless, these metabolic inhibi-tors were used in mitochondrial studies bychoosing carefully the concentrations thataffect only mitochondrial respiration but notchloroplast reactions (Igamberdiev et al.,1997a,b; Padmasree and Raghavendra,1999a,b,c).
The respiratory measurements often uti-lize the Clark-type oxygen electrode, whichmonitors only the net changes in the O2
levels (caused by both consumption of O2 inrespiration and evolution of O2 in photosyn-thesis). It is desirable that these two pro-cesses be monitored seperately, so as tomake precise measurements of photosyn-thesis or respiration. Mass spectrophotom-eter, which can monitor 18/16O2 or 13/12CO2,has been extremely useful for not only dis-tinguishing between photosynthesis and res-piration (Avelange et al., 1991) but also tomake quantitative measurements of alterna-tive pathway activity (Robinson et al., 1995).
The studies using inhibitors can becomplemented by experiments involvingmutants or transgenic plants, with an alteredpattern of proteins/enzymes related to chlo-roplasts, mitochondria, and peroxisomes.Extensive studies are made on transgenicplants with overexpression or (antisense)depression of enzymes such as triose-P de-hydrogenase, rubisco, activase, rubisco orproteins such as triose-P-phosphate-translocator (Vivekanandan and Saralabai,1997; Heineke, 1998; Sharkey, 1998; Huber,1998; Flügge, 2000; Häusler et al., 2000).Similarly, mutants or transgenics with al-tered levels of invertase or sucrose synthaseor ADP-glucose pyrophosphorylase or PRKor FBPase or glutamine synthetase orglutamate synthase are available (Häusler etal., 1994; Heineke, 1998; Paul et al., 2000).In contrast, only a few studies are made onmutants/transgenics with altered respiratorycharacteristics (Vanlerberghe et al., 1994;Hiser et al., 1996; Gutierres et al., 1997;
Igamberdiev et al., 2001). In an interestingrecent study, Sabar et al. (2000) used themale sterile mutants of Nicotiana sylvestristo study some aspects of respiration andphotosynthesis. So far, no studies have beenreported on the chloroplast-mitochondriainteractions in suitable transgenic plants.
The plant mitochondria have an uniquesystem of two different types of oxidativeelectron transport-cytochrome pathway andthe alternative pathway. Being a major routefor ATP formation in mitochondria, theimportance of cytochrome pathway is un-questionable and is obvious. However, thephysiological importance of alternative path-way is not completely understood. Oxida-tive electron transport in mitochondria oc-curs predominantly through alternativepathway during glycine oxidation in mito-chondria or LEDR in barley protoplasts(Igamberdiev et al., 1997a,b). This phenom-enon has to be analyzed further preferablyby employing tools other than the metabolicinhibitors. Further, the role of alternativepathway in optimizing chloroplast functionalso has to be studied under varied environ-mental conditions, such as light intensity,temperature, and stress conditions. The re-sults would be quite exciting and wouldhelp us understand not only interorganelleinteraction but also the alternative pathwayitself, which is unique to plant mitochon-dria.
The strong interaction between chlo-roplasts, mitochondria, peroxisomes, andcytosol is possible only when there is aneffecient cross-talk between these or-ganelles. Obviously, metabolite move-ment is an important factor or signal.However, it is possible that there are othersignals. Further work is necessary to iden-tify and establish the importance of dif-ferent signals between the organelles.Among the possibilities are cytosolic pH,phosphate status, and even the superox-ide radicals.
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The major advantage of the interorganelleinteraction appears to be optimization of theirfunction and protection from any damagedue to the unfavorable factors. For example,the chloroplasts are protected from gettingoverreduced. It is quite likely that mitochon-dria are also prevented from gettingoveroxidized. However, not much informa-tion is available pertaining to protection ofmitochondria.
On exposure to supra-optimal light andlikely photoinhibition, the mitochondria(along with peroxisomes) rescue the chloro-plasts by dissipating their excess reducedequivalents (Saradadevi and Raghavendra,1992; Hurry et al., 1998; Padmasree andRaghavendra, 2000; Gardeström et al.,2001). It would be of great interest andexciting to examine the interaction betweenthe different organelles, particularly chloro-plasts, mitochondria, and peroxisomes andthe consequences on metabolic regulationwhen the plant is subjected to other stressconditions such as temperature or water.
ACKNOWLEDGMENTS
Work in our laboratory on photosynthe-sis and respiration in mesophyll and guardcell protoplasts was supported by a grant(No. SP/SO/A-12/98) from Department ofScience and Technology, New Delhi toA.S.R. K.P. is a recipient of ResearchAssociateship and L.P. holds a Senior Re-search Fellowship, both from the Council ofScientific and Industrial Research, New Delhi.
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