Chlororespiratory reduction 6 is a novel factor required for accumulation of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

The chloroplast NAD(P)H dehydrogenase (NDH) complex is involved in photosystem I cyclic electron transport and chlororespiration in higher plants. An Arabidopsis (Arabidopsis thaliana) chlororespiratory reduction 6 (crr6) mutant lacking NDH activity was identified by means of chlorophyll fluorescence imaging. Accumulation of the NDH complex was impaired in crr6. Physiological characterization of photosynthetic electron transport indicated the specific defect of the NDH complex in crr6. In contrast to the CRR7 protein that was recently identified as a potential novel subunit of the NDH complex by means of the same screening, the CRR6 protein was stable under the crr2 mutant background in which the NDH complex does not accumulate. The CRR6 gene (At2g47910) encodes a novel protein without any known motif. Although CRR6 does not have any transmembrane domains, it is localized in the thylakoid membrane fraction of the chloroplast. CRR6 is conserved in phototrophs, including cyanobacteria, from which the chloroplast NDH complex has evolutionally originated, but not in Chlamydomonas reinhardtii, in which the NDH complex is absent. We believe that CRR6 is a novel specific factor for the assembly or stabilization of the NDH complex.     

Dihydrodipicolinate reductase-like protein, CRR1, is essential for chloroplast NAD(P)H dehydrogenase in Arabidopsis

Chloroplast NAD(P)H dehydrogenase (NDH) is a homolog of the bacterial NADH dehydrogenase NDH-1 and is involved in cyclic electron transport around photosystem I. In higher plants, 14 subunits of the NDH complex have been identified. The subunit that contains the electron donor-binding site or an electron donor to NDH has not been determined. Arabidopsis crr1 (chlororespiratory reduction 1) mutants were isolated by chlorophyll fluorescence imaging on the basis of their lack of NDH activity. CRR1 is homologous to dihydrodipicolinate reductase (DHPR), which functions in a lysine biosynthesis pathway. However, the dihydrodipicolinate-binding motif was not conserved in CRR1, and the crr1 defect was specific to accumulation of the NDH complex, implying that CRR1 is not involved in lysine biosynthesis in Arabidopsis. Similarly to other nuclear-encoded genes for NDH subunits, CRR1 was expressed only in photosynthetic tissue. CRR1 contained a NAD(P)H-binding motif and was a candidate electron donor-binding subunit of the NDH complex. However, CRR1 was detected in the stroma but not in the thylakoid membranes, where the NDH complex is localized. Furthermore, CRR1 was stable in crr2-2 lacking the NDH complex. These results suggest that CRR1 is involved in biogenesis or stabilization of the NDH complex, possibly via the reduction of an unknown substrate.     

A nucleus-encoded factor, CRR2, is essential for the expression of chloroplast ndhB in Arabidopsis

The chloroplast NDH complex, NAD(P)H dehydrogenase, reduces the plastoquinone pool non-photochemically and is involved in cyclic electron flow around photosystem I (PSI). A transient increase in chlorophyll fluorescence after turning off actinic light is a result of NDH activity. We focused on this subtle change in chlorophyll fluorescence to isolate nuclear mutants affected in chloroplast NDH activity in Arabidopsis by using chlorophyll fluorescence imaging. crr2-1 and crr2-2 (chlororespiratory reduction) are recessive mutant alleles in which accumulation of the NDH complex is impaired. Except for the defect in NDH activity, photosynthetic electron transport was unaffected. CRR2 encodes a member of the plant combinatorial and modular protein (PCMP) family consisting of more than 200 genes in Arabidopsis. CRR2 functions in the intergenic processing of chloroplast RNA between rps7 and ndhB, which is possibly essential for ndhB translation. We have determined the function of a PCMP family member, indicating that the family is closely related to pentatrico-peptide PPR proteins involved in the maturation steps of organellar RNA.     

Contribution of NDH-dependent cyclic electron transport around photosystem I to the generation of proton motive force in the weak mutant allele of pgr5

In angiosperms, cyclic electron transport (CET) around photosystem I (PSI) consists of two pathways, depending on PGR5/PGRL1 proteins and the chloroplast NDH complex. In single mutants defective in chloroplast NDH, photosynthetic electron transport is only slightly affected at low light intensity, but in double mutants impaired in both CET pathways photosynthesis and plant growth are severely affected. The question is whether this strong mutant phenotype observed in double mutants can be simply explained by the additive effect of defects in both CET pathways. In this study, we used the weak mutant allele of pgr5-2 for the background of double mutants to avoid possible problems caused by the secondary effects due to the strong mutant phenotype. In two double mutants, crr2-2 pgr5-2 and ndhs-1 pgr5-2, the plant growth was unaffected and linear electron transport was only slightly affected. However, NPQ induction was more severely impaired in the double mutants than in the pgr5-2 single mutant. A similar trend was observed in the size of the proton motive force. Despite the slight reduction in photosystem II parameters, PSI parameters were severely affected in the pgr5-2 single mutant, the phenotype that was further enhanced by adding the NDH defects. Despite the lack of ?pH-dependent regulation at the cytochrome b₆f complex (donor-side regulation of PSI), the plastoquinone pool was more reduced in the double mutants than in the pgr5-2 single mutants. This phenotype suggests that both PGR5/PGRL1- and NDH-dependent CET contribute to supply sufficient acceptors from PSI by balancing the ATP/NADPH production ratio.     

Rubisco activase is a key regulator of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature

The role of Rubisco activase in steady-state and non-steady-state photosynthesis was analyzed in wild-type (Oryza sativa) and transgenic rice that expressed different amounts of Rubisco activase. Below 25°C, the Rubisco activation state and steady-state photosynthesis were only affected when Rubisco activase was reduced by more than 70%. However, at 40°C, smaller reductions in Rubisco activase content were linked to a reduced Rubisco activation state and steady-state photosynthesis. As a result, overexpression of maize Rubisco activase in rice did not lead to an increase of the Rubisco activation state, nor to an increase in photosynthetic rate below 25°C, but had a small stimulatory effect at 40°C. On the other hand, the rate at which photosynthesis approached the steady state following an increase in light intensity was rapid in Rubisco activase-overexpressing plants, intermediate in the wild-type, and slowest in antisense plants at any leaf temperature. In Rubisco activase-overexpressing plants, Rubisco activation state at low light was maintained at higher levels than in the wild-type. Thus, rapid regulation by Rubisco activase following an increase in light intensity and/or maintenance of a high Rubisco activation state at low light would result in a rapid increase in Rubisco activation state and photosynthetic rate following an increase in light intensity. It is concluded that Rubisco activase plays an important role in the regulation of non-steady-state photosynthesis at any leaf temperature and, to a lesser extent, of steady-state photosynthesis at high temperature.     

Identification of a novel protein, CRR7, required for the stabilization of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

An Arabidopsis thaliana mutant, crr7 (chlororespiratory reduction), was isolated using chlorophyll fluorescence imaging to detect reduced activity in NAD(P)H dehydrogenase (NDH). The chloroplast NDH complex is considered to have originated from cyanobacteria in which the NDH complex is involved in respiration, photosystem I (PSI) cyclic electron transport and CO2 uptake. In higher plants the NDH complex functions in PSI cyclic electron transport within the chloroplast. Despite exhaustive biochemical approaches, the entire subunit composition of the NDH complex is unclear in both cyanobacteria and chloroplasts. In crr7 accumulation of the NDH complex was specifically impaired. In vivo analysis of electron transport supported the specific loss of the NDH complex in crr7. CRR7 (At5g39210) encodes a protein of 156 amino acids, including a putative plastid target signal, and does not contain any known motifs. In contrast to CRR2 and CRR4, involved in the expression of chloroplast ndh genes, CRR7 is conserved in cyanobacterial genomes. Although CRR7 did not contain any transmembrane domains, it localized to the membrane fraction of the chloroplast. CRR7 was unstable in the crr2-2 mutant background, in which the expression of ndhB was impaired. These results strongly suggest that CRR7 is a novel subunit of the chloroplast NDH complex.     

CRR23/NdhL is a subunit of the chloroplast NAD(P)H dehydrogenase complex in Arabidopsis

The chloroplast NAD(P)H dehydrogenase (NDH) complex functions in PSI cyclic and chlororespiratory electron transport in higher plants. Eleven plastid-encoded and three nuclear-encoded subunits have been identified so far, but the entire subunit composition, especially of the putative electron donor-binding module, is unclear. We isolated Arabidopsis thaliana crr23 (chlororespiratory reduction) mutants lacking NDH activity according to the absence of a transient increase in Chl fluorescence after actinic light illumination. Although CRR23 shows similarity to the NdhL subunit of cyanobacterial NDH-1, it has three transmembrane domains rather than the two in cyanobacterial NdhL. Unlike cyanobacterial NdhL, CRR23 is essential for stabilizing the NDH complex, which in turn is required for the accumulation of CRR23. Furthermore, CRR23 and NdhH, a subunit of chloroplast NDH, co-localized in blue-native gel. All the results indicate that CRR23 is an ortholog of cyanobacterial ndhL in Arabidopsis, despite its diversity of structure and function.     

Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response

Besides major photosynthetic complexes of oxygenic photosynthesis, new electron carriers have been identified in thylakoid membranes of higher plant chloroplasts. These minor components, located in the stroma lamellae, include a plastidial NAD(P)H dehydrogenase (NDH) complex and a plastid terminal plastoquinone oxidase (PTOX). The NDH complex, by reducing plastoquinones (PQs), participates in one of the two electron transfer pathways operating around photosystem I (PSI), the other likely involving a still uncharacterized ferredoxin-plastoquinone reductase (FQR) and the newly discovered PGR5. The existence of a complex network of mechanisms regulating expression and activity of the NDH complex, and the presence of higher amounts of NDH complex and PTOX in response to environmental stress conditions the phenotype of mutants, indicate that these components likely play a role in the acclimation of photosynthesis to changing environmental conditions. Based on recently published data, we propose that the NDH-dependent cyclic pathway around PSI participates to the ATP supply in conditions of high ATP demand (such as high temperature or water limitation) and together with PTOX regulates cyclic electron transfer activity by tuning the redox state of intersystem electron carriers. In response to severe stress conditions, PTOX associated to the NDH and/or the PGR5 pathway may also limit electron pressure on PSI acceptor and prevent PSI photoinhibition.     

Chloroplastic NAD(P)H dehydrogenase complex and cyclic electron transport around photosystem I

Recent molecular genetics studies have revealed that cyclic electron transport around photosystem I is essential for normal photosynthesis and growth of plants. Chloroplastic NAD(P)H dehydorgenase (NDH) complex, a homologue of the complex I in respiratory electron transport, is involved in one of two cyclic pathways. Recent studies on the function and structure of the NDH complex are reviewed.     

Probing the FQR and NDH activities involved in cyclic electron transport around Photosystem I by the 'afterglow' luminescence

Far-red illumination of plant leaves for a few seconds induces a delayed luminescence rise, or afterglow, that can be measured with the thermoluminescence technique as a sharp band peaking at around 40-45 degrees C. The afterglow band is attributable to a heat-induced electron flow from the stroma to the plastoquinone pool and the PSII centers. Using various Arabidopsis and tobacco mutants, we show here that the electron fluxes reflected by the afterglow luminescence follow the pathways of cyclic electron transport around PSI. In tobacco, the afterglow signal relied mainly on the ferredoxin-quinone oxidoreductase (FQR) activity while the predominant pathway responsible for the afterglow in Arabidopsis involved the NAD(P)H dehydrogenase (NDH) complex. The peak temperature T(m) of the afterglow band varied markedly with the light conditions prevailing before the TL measurements, from around 30 degrees C to 45 degrees C in Arabidopsis. These photoinduced changes in Tm followed the same kinetics and responded to the same light stimuli as the state 1-state 2 transitions. PSII-exciting light (leading to state 2) induced a downward shift while preillumination with far-red light (inducing state 1) caused an upward shift. However, the light-induced downshift was strongly inhibited in NDH-deficient Arabidopsis mutants and the upward shift was cancelled in plants durably acclimated to high light, which can perform normal state transitions. Taken together, our results suggest that the peak temperature of the afterglow band is indicative of regulatory processes affecting electron donation to the PQ pool which could involve phosphorylation of NDH. The afterglow thermoluminescence band provides a new and simple tool to investigate the cyclic electron transfer pathways and to study their regulation in vivo.     

Involvement of chlororespiration in chilling stress in the tropical species Spathiphyllum wallisii

Spathiphyllum wallisii plants were used to study the effect of chilling stress under high illumination on photosynthesis and chlororespiration. Leaves showed different responses that depended on root temperature. When stem, but not root, was chilled, photosystem II (PSII) was strongly photoinhibited. However, when the whole plant was chilled, the maximal quantum yield of PSII decreased only slightly below the normal values and cyclic electron transport was stimulated. Changes were also observed in the chlororespiration enzymes and PGR5. In whole plants chilled under high illumination, the amounts of NADH dehydrogenase (NDH) complex and plastid terminal oxidase (PTOX) remained similar to control and increased when only stem was chilled. In contrast, the amount of PGR5 polypeptide was higher in plants when both root and stem were chilled than in plants in which only stem was chilled. The results indicated that the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when the whole plant is chilled under high light, and that another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PSII activity is strongly photoinhibited in plants in which only stem is chilled, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.     

Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-dehydrogenase complex to cyclic electron flow around photosystem I

Tobacco (Nicotiana tabacum var Petit Havana) ndhB-inactivated mutants (ndhB-) obtained by plastid transformation (E.M. Horvath, S.O. Peter, T. Jo?t, D. Rumeau, L. Cournac, G.V. Horvath, T.A. Kavanagh, C. Sch?fer, G. Peltier, P. MedgyesyHorvath [2000] Plant Physiol 123: 1337-1350) were used to study the role of the NADH-dehydrogenase complex (NDH) during photosynthesis and particularly the involvement of this complex in cyclic electron flow around photosystem I (PSI). Photosynthetic activity was determined on leaf discs by measuring CO2 exchange and chlorophyll fluorescence quenchings during a dark-to-light transition. In the absence of treatment, both non-photochemical and photochemical fluorescence quenchings were similar in ndhB- and wild type (WT). When leaf discs were treated with 5 microM antimycin A, an inhibitor of cyclic electron flow around PSI, both quenchings were strongly affected. At steady state, maximum photosynthetic electron transport activity was inhibited by 20% in WT and by 50% in ndhB-. Under non-photorespiratory conditions (2% O2, 2,500 microL x L(-1) CO2), antimycin A had no effect on photosynthetic activity of WT, whereas a 30% inhibition was observed both on quantum yield of photosynthesis assayed by chlorophyll fluorescence and on CO2 assimilation in ndhB-. The effect of antimycin A on ndhB- could not be mimicked by myxothiazol, an inhibitor of the mitochondrial cytochrome bc1 complex, therefore showing that it is not related to an inhibition of the mitochondrial electron transport chain but rather to an inhibition of cyclic electron flow around PSI. We conclude to the existence of two different pathways of cyclic electron flow operating around PSI in higher plant chloroplasts. One of these pathways, sensitive to antimycin A, probably involves ferredoxin plastoquinone reductase, whereas the other involves the NDH complex. The absence of visible phenotype in ndhB- plants under normal conditions is explained by the complement of these two pathways in the supply of extra-ATP for photosynthesis.     

NDF6: a thylakoid protein specific to terrestrial plants is essential for activity of chloroplastic NAD(P)H dehydrogenase in Arabidopsis

NAD(P)H dehydrogenase (NDH) is a homolog of respiratory complex I and mediates one of the two pathways of cyclic electron flow around PSI (CEF I). Although 15 ndh subunits have been identified in the chloroplastic and nuclear genomes of higher plants, no electron accepter subunits have been identified to date. To identify the missing chloroplastic NDH subunits, we undertook an in silico approach based on co-expression analysis. In this report, we characterized the novel gene NDF6 (NDH-dependent flow 6; At1g18730) which encodes a protein that is essential for NDH activity. NDF6 has one transmembrane domain and is localized in the thylakoid membrane fraction. Homologous proteins of NDF6 were identified in the genomes of terrestrial plants; however, no homologs have been found in cyanobacteria, which are thought to be the origin of chloroplasts and have a minimal NDH complex unit. NDF6 is unstable in ndhB-impaired or disrupted mutants of higher plants in which the chloroplastic NDH complex is thought to be degraded. These results suggest that NDF6 is a novel subunit of chloroplastic NDH that was added to terrestrial plants during evolution.     

Electron transport activities of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> mutants with impaired chloroplastic NAD(P)H dehydrogenase

The activities of electron transport are compared between wild-type Arabidopsis and two Arabidopsis mutants deficient for the chloroplastic NAD(P)H dehydrogenase (NDH) which catalyzes cyclic electron transport around photosystem I. The quantum yield of photosystem II and the degree of non-photochemical quenching of chlorophyll fluorescence were of similar levels in the two NDH-deficient mutants and the wild type under non-stressed standard growth conditions. Stromal over-reduction was induced in Arabidopsis NDH mutants with high light treatment, as is the case in tobacco NDH mutants. However, unlike tobacco mutants, photoinhibition was not observed in the Arabidopsis NDH mutants.     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

After incubation at 42°C for more than 48 h, brown damages occurred on the stems of tobacco (Nicotiana tabacum L.) ndhC-ndhK-ndhJ deletion mutant (ΔndhCKJ), followed by wilt of the leaves, while less the phenotype was found in its wild type (WT). Analysis of the kinetics of post-illumination rise in chlorophyll fluorescence indicated that the PSI cyclic electron flow and the chlororespiration mediated by NAD(P)H dehydrogenase (NDH) was significantly enhanced in WT under the high temperature. After leaf disks were treated with methyl viologen (MV), photosynthetic apparatus of ΔndhCKJ exhibited more severe photo-oxidative damage, even bleaching of chlorophyll. Analysis of P700 oxidation and reduction showed that the NDH mediated cyclic electron flow probably functioned as an electron competitor with Mehler reaction, to reduce the accumulation of reactive oxygen species (ROS). When leaf disks were heat stressed at 42°C for 6 h, the photochemical activity declined more markedly in ΔndhCKJ than in WT, accompanied with more evident decrease in the amount of soluble Rubisco activase. In addition, the slow phase of millisecond-delayed light emission (ms-DLE) of chlorophyll fluorescence indicated that NDH was involved in the building-up of transthylakoid proton gradient (ΔpH), while the consumption of ΔpH was highly inhibited in ΔndhCKJ after heat stress. Based on the results, we supposed that the cyclic electron flow mediated by NDH could be stimulated under the heat stressed conditions, to divert excess electrons via chlororespiration pathway, and sustain CO₂ assimilation by providing extra ΔpH, thus reducing the photooxidative damage.     

The role of chloroplast NAD(P)H dehydrogenase in protection of tobacco plant against heat stress

The environmental temperature is one of the mainfactors affecting plant growth and development. Insummer, plants are frequently influenced by hightemperature. In recent years, global temperature wasremarkably elevated accompanied with the climaticchanges,…     

Probing the FQR and NDH activities involved in cyclic electron transport around Photosystem I by the ‘afterglow’ luminescence

Far-red illumination of plant leaves for a few seconds induces a delayed luminescence rise, or afterglow, that can be measured with the thermoluminescence technique as a sharp band peaking at around 40-45 °C. The afterglow band is attributable to a heat-induced electron flow from the stroma to the plastoquinone pool and the PSII centers. Using various Arabidopsis and tobacco mutants, we show here that the electron fluxes reflected by the afterglow luminescence follow the pathways of cyclic electron transport around PSI. In tobacco, the afterglow signal relied mainly on the ferredoxin-quinone oxidoreductase (FQR) activity while the predominant pathway responsible for the afterglow in Arabidopsis involved the NAD(P)H dehydrogenase (NDH) complex. The peak temperature T_m of the afterglow band varied markedly with the light conditions prevailing before the TL measurements, from around 30 °C to 45 °C in Arabidopsis. These photoinduced changes in T_m followed the same kinetics and responded to the same light stimuli as the state 1-state 2 transitions. PSII-exciting light (leading to state 2) induced a downward shift while preillumination with far-red light (inducing state 1) caused an upward shift. However, the light-induced downshift was strongly inhibited in NDH-deficient Arabidopsis mutants and the upward shift was cancelled in plants durably acclimated to high light, which can perform normal state transitions. Taken together, our results suggest that the peak temperature of the afterglow band is indicative of regulatory processes affecting electron donation to the PQ pool which could involve phosphorylation of NDH. The afterglow thermoluminescence band provides a new and simple tool to investigate the cyclic electron transfer pathways and to study their regulation in vivo.     

The Effects of Cold Stress on Photosynthesis in Hibiscus Plants

The present work studies the effects of cold on photosynthesis, as well as the involvement in the chilling stress of chlororespiratory enzymes and ferredoxin-mediated cyclic electron flow, in illuminated plants of Hibiscus rosa-sinensis. Plants were sensitive to cold stress, as indicated by a reduction in the photochemistry efficiency of PSII and in the capacity for electron transport. However, the susceptibility of leaves to cold may be modified by root temperature. When the stem, but not roots, was chilled, the quantum yield of PSII and the relative electron transport rates were much lower than when the whole plant, root and stem, was chilled at 10°C. Additionally, when the whole plant was cooled, both the activity of electron donation by NADPH and ferredoxin to plastoquinone and the amount of PGR5 polypeptide, an essential component of the cyclic electron flow around PSI, increased, suggesting that in these conditions cyclic electron flow helps protect photosystems. However, when the stem, but not the root, was cooled cyclic electron flow did not increase and PSII was damaged as a result of insufficient dissipation of the excess light energy. In contrast, the chlororespiratory enzymes (NDH complex and PTOX) remained similar to control when the whole plant was cooled, but increased when only the stem was cooled, suggesting the involvement of chlororespiration in the response to chilling stress when other pathways, such as cyclic electron flow around PSI, are insufficient to protect PSII.     

A balanced PGR5 level is required for chloroplast development and optimum operation of cyclic electron transport around photosystem I

PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of non-photochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis.     

Influence of light intensity during growth on photosynthesis and activity of several key photosynthetic enzymes in a C4 plant (Zea mays)

Maize ( Zea mays L. Hybrid Sweet Corn, Royal Crest), a C₄ plant, was grown under different light regimes, after which the rate of photosynthesis and activities of several photosynthetic enzymes (per unit leaf chlorophyll) were measured at different light intensities. Plants were grown outdoors under direct sunlight or 23% of direct sunlight, and in growth chambers at photosynthetic photon flux densities of about 20% and 8% of direct sunlight. The plants grown under direct sunlight had a higher light compensation point than plants grown under lower light. At a light intensity about 25% of direct sunlight, plants from all growth regimes had a similar rate of photosynthesis. Under saturating levels of light the plants grown under direct sunlight had a substantially higher rate of photosynthesis than plants grown under the lower light regimes. The higher photosynthetic capacity in the plants grown under direct sunlight was accompanied by an increased activity of several photosynthetic enzymes and in the amount of the soluble protein in the leaf. Among five photosynthetic enzymes examined, RuBP carboxylase (EC 4.1.1.39) and pyruvate, Pi dikinase (EC 2.7.9.1) were generally just sufficient to account for rates of photosynthesis under saturating light; thus, these may be rate limiting enzymes in C₄ photosynthesis. Pyruvate, Pi dikinase and NADP-malate dehydrogenase (EC 1.1.1.82) were the only enzymes examined which were light activated and increased in activity with increasing light intensity. In the low light grown plants the activity of pyruvate, Pi dikinase closely paralleled the photosynthetic rate measured under different light levels. With the plants grown under direct sunlight, as light intensity was increased the activation of pyruvate, Pi dikinase and NADP⁺-malate dehydrogenase proceeded more rapidly than photosynthesis.