Iron, sulfur, and chlorophyll deficiencies: A need for an integrative approach in plant physiology

Green plants deficient in nitrogen, sulfur, or iron develop a similar yellow coloration. In each case, the yellow coloration is accompanied by a lowered chlorophyll concentration. This review attempts to collate some of the biochemical information concerning these three seemingly diverse nutritive deficiencies and bares a need for a more integrative approach to plant physiology. The biochemical and biological roles of nitrogen, sulfur and iron in living systems are examined, with emphasis on sulfur and iron. Mechanistically, iron and/or sulfur are highly reactive components of many enzymes. Indeed, iron and sulfur sometimes form Fe₂S₂, Fe₃S₄, or Fe₄S₄ clusters which are very active electron transfer agents. Recently, iron‐sulfur clusters have been reported to serve as sensors of oxidative stress, to couple photosynthesis with several metabolic pathways, to participate in the reduction of sulfite and nitrite, and to participate in regulation of gene expression. Thus, there are several mechanisms by which a deficiency of nitrogen, sulfur, or iron could produce the same low‐chlorophyll, yellow phenotype in plants. Unless the interactions and coordination of the various pathways connected to chlorophyll synthesis are elucidated, it is unlikely that we will select the quickest and most direct path to plant improvement.     

Molecular dissection of photosystem I in higher plants: topology, structure and function

Photosystem I catalyses the light driven electron transfer from plastocyanin/cytochrome c ₆ on the lumenal side of the thylakoid membrane to ferredoxin/flavodoxin at the stromal side. Photosystem I of higher plants consists of 18 different protein subunits. Fourteen of these make up the chlorophyll a -containing core, which also contains the cofactors involved in the electron transfer reactions, and four make up the peripheral chlorophyll a / b -containing antenna. Arabidopsis plants devoid of the nuclear-encoded photosystem I subunits have been obtained either by different suppression techniques or by insertional knock-out of the genes. This has allowed a detailed analysis of the role and function of the individual subunits. This review is focused on recent developments in the role of the individual subunit in the structure and function of photosystem I of higher plants.     

Chloroplast structure in normal and pigment-deficient soybeans grown in continuous red or far-red light

Clark L1, a normal green soybean [ Glycine max (L.) Merrill] and Clark y₉y₉, a backross-developed isoline exhibiting pigment deficiency, were grown under continuous red (11 W m⁻² and far-red (9 W m⁻²) light. Chloroplast thylakoids from the unifoliolate leaf (9–10 days old) were isolated and analyzed for pigments, pigment-protein, membrane polypeptides, electron transport and ultrastructural differences. Chloroplasts of soybean plants grown under far-red light have decreased chlorophyll a to chlorophyll b ratio, increased light-harvesting complexes, and grana structure with few stroma-type thylakoids. Photosystem II/photosystem I ratios (PSII/PSI) are higher in far-red due to decreased synthesis of PSI reaction center and/or less antenna associated with PSI.     

A unique photosystem I reaction center from a chlorophyll d-containing cyanobacterium Acaryochloris marina

Photosystem I (PSI) is a large protein supercomplex that catalyzes the light-dependent oxidation of plastocyanin (or cytochrome c₆) and the reduction of ferredoxin. This catalytic reaction is realized by a transmembrane electron transfer chain consisting of primary electron donor (a special chlorophyll (Chl) pair) and electron acceptors A₀, A₁, and three Fe₄S₄ clusters, F_X, F_A, and F_B. Here we report the PSI structure from a Chl d-dominated cyanobacterium Acaryochloris marina at 3.3 Å resolution obtained by single-particle cryo-electron microscopy. The A. marina PSI exists as a trimer with three identical monomers. Surprisingly, the structure reveals a unique composition of electron transfer chain in which the primary electron acceptor A₀ is composed of two pheophytin a rather than Chl a found in any other well-known PSI structures. A novel subunit Psa27 is observed in the A. marina PSI structure. In addition, 77 Chls, 13 α-carotenes, two phylloquinones, three Fe-S clusters, two phosphatidyl glycerols, and one monogalactosyl-diglyceride were identified in each PSI monomer. Our results provide a structural basis for deciphering the mechanism of photosynthesis in a PSI complex with Chl d as the dominating pigments and absorbing far-red light.     

Photosystem I Is an Early Target of Photoinhibition in Barley Illuminated at Chilling Temperatures

Light-induced damage to photosystem I (PSI) was studied during low-light illumination of barley (Hordeum vulgare L.) at chilling temperatures. A 4-h illumination period induced a significant inactivation of PSI electron transport activity. Flash-induced P700 absorption decay measurements revealed progressive damage to (a) the iron-sulfur clusters F_A and F_B, (b) the iron-sulfur clusters F_A, F_B, and F_X, and (c) the phylloquinone A₁ and the chlorophyll A₀ or P700 of the PSI electron acceptor chain. Light-induced PSI damage was also evidenced by partial degradation of the PSI-A and PSI-B proteins and was correlated with the appearance of smaller proteins. Aggravated photodamage was observed upon illumination of barley leaves infiltrated with KCN, which inhibits Cu,Zn-superoxide dismutase and ascorbate peroxidase. This indicates that the photodamage of PSI in barley observed during low-light illumination at chilling temperatures arises because the defense against active oxygen species by active oxygen-scavenging enzymes is insufficient at these specific conditions. The data obtained demonstrate that photoinhibition of PSI at chilling temperatures is an important phenomenon in a cold-tolerant plant species.     

Adaptability of tomato and pepper leaves to changes in photoperiod: Effects on the composition and function of the thylakoid membrane

The adaptability of the thylakoid membrane to extended photoperiod (from natural to 24 h) was studied using a photoperiod-sensitive species ( Lycopersicon esculentum Mill. cv. Trend) and a non-photoperiod-sensitive species ( Capsicum annuum L. cv. Delphin). Our results have shown that thylakoid membranes of both species adapt to an extended photoperiod by increasing their photosystem II to photosystem I ratio (PSII/PSI) in order to provide a more balanced energy distribution between both photosystems to improve quantum yield. In tomato plants, these results correspond with a lower chlorophyll (Chl) a/b ratio, a decrease in Chl associated with PSI light-harvesting chlorophyll a/b protein complexes and with an increase in Chl associated with PSII light-harvesting chlorophyll a/b protein complexes. In spite of these changes, the electron transport capacity through PSII and PSI per unit of Chl and the light saturation point of PSII remained unchanged. The inability of tomato plants to use supplemental light for an extended photoperiod is not the result of photoinhibitory conditions. In pepper plants a significant increase in electron transport capacity and in the light saturation point of PSII was found. There was a significant increase in CO₂ assimilation when the light period was increased from 12 to 24 h. In contrast to tomato, pepper plants adapt to a 24-h photoperiod by increasing their carboxylation capacity which is accompanied by an increase in electron transport capacity and the light saturation point.     

Iron-sulfur clusters: Formation,perturbation, and physiological functions

Iron-sulfur proteins occur in all life forms. Ferredoxins and Rieske proteins each contain a (2Fe2S) cluster whereas photosystem I (PSI) contains three (4Fe4S) clusters. Essential enzymes such as sulfite reductase, nitrite reductase, nitrogenase, glutamate synthase, aconitase, succinate dehydrogenase, ferredoxin/thioredoxin reductase, as well as many other vital proteins, each contain a (4Fe4S) cluster. Iron-sulfur clusters are formed enzymatically from cysteinyl-sulfur and ferritin-sequestered iron. Many iron-sulfur clusters are inactivated by O₂ and/or reactive oxygen species (ROS) such as O₂^•−. Perhaps 0.1 % of the electrons passing through either the mitochondrial electron transport chain or PSI result in the formation of O₂^•−. Many plant stresses increase ROS formation, which subsequently may perturb iron-sulfur clusters. Plants have evolved three different superoxide dismutases (SODs) to control the internal concentrations of harmful ROS. Possible roles of functional and non-functional iron-sulfur clusters in the coordination of metabolic activities of stressed and non-stressed plants are discussed.     

Effect of carbon dioxide enrichment on chlorophyll content, starch content and starch grain structure in Trifolium subterraneum leaves

Trifolium subterraneum (cv. Dinninup) responds to enriched atmospheric CO₂ in a manner similar to that described by Madsen (1968 and 1976) for tomato. In immature leaves, the total chlorophyll content per unit dry weight and the chlorophyll a:b ratio are significantly lower in plants grown at 0.10 vol% CO₂. Although fully expanded mature leaves partially overcome the deficit in chlorophyll content, the chlorophyll a:b ratio remains substantially lower in these high CO₂ grown plants. The large amount of starch accumulated as irregularly shaped grains appears to disrupt normal chloroplast structure in clover plants grown in enriched atmospheric CO₂. These results indicate the chlorotic appearance of leaves from high CO₂ grown clover plants is due to a decrease in chlorophyll content per dry weight possibly resulting from large starch grains and starch accumulation altering normal chloroplast structure and function.     

Responses of Jatropha curcas seedlings to cold stress: photosynthesis-related proteins and chlorophyll fluorescence characteristics

Photosynthesis-related proteins and PSII functions of Jatropha curcas seedlings under cold stress were studied using proteomic and chlorophyll fluorescence approaches. The results of chlorophyll fluorescence measurement indicated that electron transport flux per reaction center (ET_o/RC) and performance index (PI_ABS) were relatively sensitive to low temperature, especially at early stage of cold stress. The increase in O–J phase and decrease in J–I phase of chlorophyll fluorescence transient indicated a protection mechanism of J.   curcas to photoinhibition at early stage of cold stress. Eight photosynthesis-related proteins significantly changed during cold stress were identified using liquid chromatography MS/MS. Results of correlation analyses between photosynthesis-related proteins and chlorophyll fluorescence parameters indicated that (1) ATP synthase and Rieske FeS protein were significantly correlated with electron transport of reaction center in PSII; (2) precursor for 33-kDa protein was positively correlated with fluorescence quenching of the O–J and J–I phases and PI_ABS during cold stress, which implies that it might be related to multiple process in PSII; (3) contrary correlations were found between F_J − F_o and two enzymes in the Calvin cycle, and the relations between these proteins and PSII function were unclear. The combined study using proteomic approaches and chlorophyll fluorescence measurements indicated that the early-stage (0–12 h) acclimation of PSII and the late-stage (after 24 h) H₂O₂ scavenging might be involved in the cold response mechanisms of J.   curcas seedlings.     

Heterogeneity of photosystem II reaction centers as influenced by heat treatment of barley leaves

Three functionally distinct populations of PSII reaction centers differing in the ability to keep the primary acceptors in a reduced state and to transfer electrons to PSI were estimated using chlorophyll fluorescence measurements in primary barley leaves exposed to elevated temperatures in the range of 37–51°C. The capacity of the PSII reaction centers to perform at least one light-induced charge separation was not affected by a 5-min heat treatment at temperatures up to 51°C. The first population containing Q_B-non-reducing centers corresponded to 15–20% of the total PSII activity up to 45°C. In a second population, PSII reaction centers maintained Q_A reduction under light in the presence of oxygen, but not in the presence of a strong artificial PSI electron acceptor, methyl viologen. In a third population that gradually increases from zero at 37°C to about 60% at 45°C, the PSII centers were not able to keep Q_A in the reduced state even in the presence of oxygen as the sole electron acceptor. Three electron transport pathways, the pseudocyclic one involving both PSII and PSI, the NAD(P)H-dependent pathway mediated by PSI alone after the loss of activity in some PSII centers, and the PSI-driven ferredoxin-dependent route enhanced by weakly efficient PSII centers that are able to provide only catalytic amounts of electrons, are suggested to create a proton gradient in chloroplasts of heat-stressed leaves thus protecting PSII reaction centers from photodamage.     

Paramecium Has Two Regulatory Subunits of Cyclic AMP-Dependent Protein Kinase, One Unique to Cilia

ABSTRACT. The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R₄₄) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R₄₄ were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R₄₈) weakly recognized by one of several monoclonal antibodies against R₄₄, but not recognized by an anti-R₄₄ polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R₄₈ and a 70-kDa form containing C and R₄₄. In the large enzyme, R₄₈ was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R₄₄ The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R₄₈ were retained by the column and eluted with 1 M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R₄₄, whereas R₄₈ is the R subunit of the large enzyme.     

Cyanobacterial NDH-1 complexes: multiplicity in function and subunit composition

In cyanobacteria, the NAD(P)H:quinone oxidoreductase (NDH-1) is involved in a variety of functions like respiration, cyclic electron flow around PSI and CO₂ uptake. Several types of NDH-1 complexes, which differ in structure and are responsible for these functions, exist in cyanobacterial membranes. This minireview is based on data obtained by reverse genetics and proteomics studies and focuses on the structural and functional differences of the two types of cyanobacterial NDH-1 complexes: NDH-1L, important for respiration and PSI cyclic electron flow, and NDH-1MS, the low-CO₂ inducible complex participating in CO₂ uptake. The NDH-1 complexes in cyanobacteria share a common NDH-1M 'core' complex and differ in the composition of the distal membrane domain composed of specific NdhD and NdhF proteins, which in complexes involved in CO₂ uptake is further associated with the hydrophilic carbon uptake (CUP) domain. At present, however, very important questions concerning the nature of catalytically active subunits that constitute the electron input device (like NADH dehydrogenase module of the eubacterial 'model' NDH-1 analogs), the substrate specificity and reaction mechanisms of cyanobacterial complexes remain unanswered and are shortly discussed here.     

Rieske Iron–Sulfur Proteins from Extremophilic Organisms

Proteins located on the outside of the membranes of organisms thriving under extreme conditions like high or low pH, or high salinity face special challenges maintaining their structural integrity. This review is focused on the Rieske iron-sulfur proteins from these organisms. Rieske proteins are essential subunits of the cytochrome bc-complexes, which are often of crucial importance for the energy metabolism of the cells. On the basis of the available data we propose strategies by which these proteins are able to stabilize their noncovalent bound cofactor and adapt to the function under extreme conditions.     

Protein-protein and protein-function relationships in Arabidopsis photosystem I: cluster analysis of PSI polypeptide levels and photosynthetic parameters in PSI mutants

In flowering plants, photosystem I (PSI) mediates electron transport across the thylakoid membrane and contains at least 14 proteins. The availability of co-suppression and/or mutant lines deficient for individual PSI polypeptides in Arabidopsis thaliana allows one to assign functions to PSI subunits. We have performed cluster analysis on an extensive set of data on PSI polypeptide levels in ten different PSI mutants. This type of analysis serves to group proteins that exhibit similar changes in amount in different genotypes, and also identifies genotypes which show similar PSI compositions. The interdependence of levels of PSI-C, -D and -E, of -H and -L, and of Lhca2 and 3, which was previously proposed based on the study of single genotypes or on cross-linking experiments, was confirmed by our analyses. In addition, the levels of the lumenal subunits F and N are found to be interdependent. The incorporation of photosynthetic parameters into the cluster analysis revealed that the level of photosynthetic state transitions correlates with the abundance of PSI-H in all 8 genotypes tested, supporting the hypothesis that PSI-H serves as a docking site for LHCII during state transitions.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

The cytochrome b6f complex. Novel aspects

The Cyt b ₆ f complex from plant chloroplasts, the green alga Chlamydomonas reinhardtii , and the thermophilic cyanobacterium, Mastigocladus laminosus , can be isolated in a highly active state, in which it is dimeric and contains one bound chlorophyll a molecule per monomeric unit. The latter feature is a distinguishing trait compared to the b ₆ f complex of bacterial photosynthesis and the respiratory chain. In contrast to the trans-membrane domains of the b ₆ f complex, and of most other integral membrane proteins, which are characterized by an a -helical structure, the p -side peripheral domains, consisting of Cyt f and the Rieske protein, have a predominantly β-strand secondary structure motif. One consequence of this motif is an extension of these polypeptides from the membrane surface. For example, the length of Cyt f is 75 Å. The heme Fe is 45 Å from the α-carbon of Arg250 at the membrane bilayer interface and, even though Cyt f may be tilted relative to the membrane plane, the heme electron transfer reactions are carried out far from the membrane surface. The presence of an internal 5 water chain, which has the properties of a proton wire, with one water H-bonded to the histidine-25 heme ligand, also suggests that the pathway of long distance H⁺ translocation traverses the extended p -side protein domain of the b ₆ f complex. A mechanism of H⁺ transfer in the chain that is coupled to the redox state of the heme, in which a proton is transferred into the chain to compensate the extra electron in the ferro-heme, is proposed.     

Role of subunits in eukaryotic Photosystem I.

Photosystem I (PSI) of eukaryotes has a number of features that distinguishes it from PSI of cyanobacteria. In plants, the PSI core has three subunits that are not found in cyanobacterial PSI. The remaining 11 subunits of the core are conserved but several of the subunits have a different role in eukaryotic PSI. A distinguishing feature of eukaryotic PSI is the membrane-imbedded peripheral antenna. Light-harvesting complex I is composed of four different subunits and is specific for PSI. Light-harvesting complex II can be associated with both PSI and PSII. Several of the core subunits interact with the peripheral antenna proteins and are important for proper function of the peripheral antenna. The review describes the role of the different subunits in eukaryotic PSI. The emphasis is on features that are different from cyanobacterial PSI.     

Relationship between mitochondrial NADH-ubiquinone reductase and a bacterial NAD-reducing hydrogenase

Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme. The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins. The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters. Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction. The mature protein is 444 amino acids long. Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16. This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN. The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal. The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN. Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3. Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits. This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.     

Carbonic anhydrase subunits of the mitochondrial NADH dehydrogenase complex (complex I) in plants

The mitochondrial nicotinamide adenine dinucleotide, reduced (NADH) dehydrogenase complex (complex I) of plants has a molecular mass of about 1000 kDa and is composed of more than 40 distinct protein subunits. About three quarter of these subunits are homologous to complex I subunits of heterotrophic eukaryotes, whereas the remaining subunits are unique to plants. Among them are three to five structurally related proteins that resemble an archaebacterial γ-type carbonic anhydrase (γCA). The γCA subunits are attached to the membrane arm of complex I on the matrix-exposed side and form an extra spherical domain. At the same time, they span the inner mitochondrial membrane and are essential for assembly of the protein complex. Expression of the genes encoding γCA subunits is reduced if plants are cultivated in the presence of elevated CO₂ concentration. The functional role of these subunits within plant mitochondria is currently unknown but might be related to photorespiration. We propose that the complex I–integrated γCAs are involved in mitochondrial HCO₃⁻ formation to allow efficient recycling of inorganic carbon for CO₂ fixation in chloroplasts under high light conditions.