Organization and expression of the mitochondrial genome in the Nicotiana sylvestris CMSII mutant.

Previous analyses suggested that the Nicotiana sylvestris CMSII mutant carried a large deletion in its mitochondrial genome. Here, we show by cosmid mapping that the deletion is 60 kb in length and contains several mitochondrial genes or ORFs, including the complex I nad7 gene. However, due to the presence of large duplications in the progenitor mitochondrial genome, the only unique gene that appears to be deleted is nad7. RNA gel blot data confirm the absence of nad7 expression, strongly suggesting that the molecular basis for the CMSII abnormal phenotype, poor growth and male sterility, is the altered complex I structure. The CMSII mitochondrial genome appears to consist essentially of one of two subgenomes resulting from recombination between direct short repeats. In the progenitor mitochondrial genome both recombination products are detected by PCR and, reciprocally, the parental fragments are detected at the substoichiometric level in the mutant. The CMSII mtDNA organization has been maintained through six sexual generations.     

Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation

Chloroplasts and mitochondria are traditionally considered to be autonomous organelles but they are not as independent as they were once thought to be. Mitochondrial metabolism, particularly the bioenergetic reactions of oxidative electron transport and phosphorylation, continue to be active in the light and are essential for sustaining photosynthetic carbon assimilation. The marked and mutually beneficial interaction between mitochondria and chloroplasts is intriguing. The key compartments within plant cells, including not only mitochondria and chloroplasts but also the peroxisomes and cytosol, appear to be in a delicate metabolic equilibrium. Disturbance of any of these compartments perturbs the metabolism of whole cell. Nevertheless, mitochondria appear to be the key players because they function during both photorespiration and dark respiration.     

The lack of mitochondrial complex I in a CMSII mutant of Nicotiana sylvestris increases photorespiration through an increased internal resistance to CO2 diffusion

The cytoplasmic male sterile II (CMSII) mutant lacking complex I of the mitochondrial electron transport chain has a lower photosynthetic activity but exhibits higher rates of excess electron transport than the wild type (WT) when grown at high light intensity. In order to examine the cause of the lower photosynthetic activity and to determine whether excess electrons are consumed by photorespiration, light, and intercellular CO(2), molar fraction (c(i)) response curves of carbon assimilation were measured at varying oxygen molar fractions. While oxygen is the major acceptor for excess electrons in CMSII and WT leaves, electron flux to photorespiration is favoured in the mutant as compared with the WT leaves. Isotopic mass spectrometry measurements showed that leaf internal conductance to CO(2) diffusion (g(m)) in mutant leaves was half that of WT leaves, thus decreasing the c(c) and favouring photorespiration in the mutant. The specificity factor of Rubisco did not differ significantly between both types of leaves. Furthermore, carbon assimilation as a function of electrons used for carboxylation processes/electrons used for oxygenation processes (J(C)/J(O)) and as a function of the calculated chloroplastic CO(2) molar fraction (c(c)) values was similar in WT and mutant leaves. Enhanced rates of photorespiration also explain the consumption of excess electrons in CMSII plants and agreed with potential ATP consumption. Furthermore, the lower initial Rubisco activity in CMSII as compared with WT leaves resulted from the lower c(c) in ambient air, since initial Rubisco activity on the basis of equal c(c) values was similar in WT and mutant leaves. The retarded growth and the lower photosynthetic activity of the mutant were largely overcome when plants were grown in high CO(2) concentrations, showing that limiting CO(2) supply for photosynthesis was a major cause of the lower growth rate and photosynthetic activity in CMSII.     

Thermal responses of Symbiodinium photosynthetic carbon assimilation

The symbiosis between hermatypic corals and their dinoflagellate endosymbionts, genus Symbiodinium, is based on carbon exchange. This symbiosis is disrupted by thermally induced coral bleaching, a stress response in which the coral host expels its algal symbionts as they become physiologically impaired. The disruption of the dissolved inorganic carbon (DIC) supply or the thermal inactivation of Rubisco have been proposed as sites of initial thermal damage that leads to the bleaching response. Symbiodinium possesses a highly unusual Form II ribulose bisphosphate carboxylase/oxygenase (Rubisco), which exhibits a lower CO₂:O₂ specificity and may be more thermally unstable than the Form I Rubiscos of other algae and land plants. Components of the CO₂ concentrating mechanism (CCM), which supplies inorganic carbon for photosynthesis, may also be temperature sensitive. Here, we examine the ability of four cultured Symbiodinium strains to acquire and fix DIC across a temperature gradient. Surprisingly, the half-saturation constant of photosynthesis with respect to DIC concentration (K _P), an index of CCM function, declined with increasing temperature in three of the four strains, indicating a greater potential for photosynthetic carbon acquisition at elevated temperatures. In the fourth strain, there was no effect of temperature on K _P. Finding no evidence for thermal inhibition of the CCM, we conclude that CCM components are not likely to be the primary sites of thermal damage. Reduced photosynthetic quantum yields, a hallmark of thermal bleaching, were observed at low DIC concentrations, leaving open the possibility that reduced inorganic carbon availability is involved in bleaching.     

A dinucleotide mutation in dihydrodipicolinate synthase of Nicotiana sylvestris leads to lysine overproduction

By applying a mutagenesis/selection procedure to obtain resistance to a lysine analog, S-(2-aminoethyl)l -cysteine (AEC), a lysine overproducing mutant in Nicotiana sylvestris was isolated. Amino acid analyses performed throughout plant development and of different organs of the N. sylvestris RAEC-1 mutant, revealed a developmental-dependent accumulation of free lysine. Lysine biosynthesis in the RAEC-1 mutant was enhanced due to a lysine feedback-desensitized dihydrodipicolinate synthase (DHDPS). Several molecular approaches were undertaken to identify the nucleotide change in the dhdps-r1 gene, the mutated gene coding for the lysine-desensitized enzyme. The enzyme was purified from wild-type plants for amino end microsequencing and 10 amino acids were identified. Using dicotyledon dhdps probes, a genomic fragment was cloned from an enriched library of DNA from the homozygote RAEC-1 mutant plant. A dhdps cDNA, putatively full-length, was isolated from a tobacco cDNA library. Nucleotide sequence analyses confirmed the presence of the previously identified amino end preceded by a chloroplast transit peptide sequence. Nucleotide sequence comparisons, enzymatic and immunological analyses revealed that the tobacco cDNA corresponds to a normal type of DHDPS, lysine feedback-regulated, and the genomic fragment to the mutated DHDPS, insensitive to lysine inhibition. Functional complementation of a DHDPS-deficient Escherichia coli strain was used as an expression system. Reconstruction between the cDNA and genomic fragment led to the production of a cDNA producing an insensitive form of DHDPS. Amino acid sequence comparisons pointed out, at position 104 from the first amino acid of the mature protein, the substitution of Asn to lleu which corresponds to a dinucleotide mutation. This change is unique to the dhdps-r1 gene when compared with the wild-type sequence. The identification of the nucleotide and amino acid change of the lysine-desensitized DHDPS from RAEC-1 plant opens new perspectives for the improvement of the nutritional value of crops and possibly to develop a new plant selectable marker.     

Contribution of Gamma amino butyric acid (GABA) to salt stress responses of Nicotiana sylvestris CMSII mutant and wild type plants

Plants accumulate high levels of Gamma amino butyric acid (GABA) in response to different environmental stresses and GABA metabolism has different functions such as osmotic and pH regulation, bypass of tricarboxylic acid cycle, and C:N balance. The cytoplasmic male sterile (CMS) II mutant of Nicotiana sylvestris has a deletion in the mitochondrial gene nad7 which encodes the NAD7 subunit of complex I which causes increased leaf respiration, impaired photosynthesis, slower growth and increased amino acid levels. In this study we aimed to elucidate the role of GABA and GABA metabolism in different genotypes of the same plant system under salt stress (100mM NaCl) in short (24h) and long (7, 14 and 21 days) terms. We have investigated the differences in leaf fresh and dry weights, relative water content, photosynthetic efficiency (F(v)/F(m)), glutamate dehydrogenase (GDH, EC 1.4.1.4) and glutamate decarboxylase (GAD, EC 4.1.1.15) enzyme activities, GABA content and GAD gene expression profiles. GDH activity showed variations in CMSII and wild type (WT) plants in the first 24h. GAD gene expression profiles were in good agreement with the GAD enzyme activity levels in CMSII and WT plants after 24h. In long-term salinity, GAD activities increased in WT but, decreased in CMSII. GABA accumulation in WT and CMSII plants in short and long term was induced by salt stress. Variations in GDH and GAD activities in relation to GABA levels were discussed and GABA metabolism has been proposed to be involved in better performance of CMSII plants under long term salinity.     

The mechanisms contributing to photosynthetic control of electron transport by carbon assimilation in leaves

Photosynthetic control describes the processes that serve to modify chloroplast membrane reactions in order to co-ordinate the synthesis of ATP and NADPH with the rate at which these metabolites can be used in carbon metabolism. At low irradiance, optimisation of the use of excitation energy is required, while at high irradiance photosynthetic control serves to dissipate excess excitation energy when the potential rate of ATP and NADPH synthesis exceed demand. The balance between pH, ATP synthesis and redox state adjusts supply to demand such that the [ATP]/[ADP] and [NADPH]/[NADP⁺] ratios are remarkably constant in steady-state conditions and modulation of electron transport occurs without extreme fluctuations in these pools.Abbreviations FBPase Fructose-1,6-bisphosphatase - PS I Photosystem I - PS II Photosystem II - Pi inorganic phosphate - PGA glycerate 3-phosphate - PQ plastoquinone - Q_A the bound quinone electron acceptor of PS II - q_P Photochemical quenching of chlorophyll fluorescence associated with the oxidation of Q_A - q_N non-photochemical quenching of chlorophyll fluorescence - q_E non-photochemical quenching associated with the high energy state of the membrane - RuBP ribulose-1,5-bisphosphate - TP triose phosphate - intrinsic quantum yield of PS II - quantum yield of electron transport - quantum yield of CO₂ assimilation     

Isolation of antibiotic resistant variants in a higher plant,Nicotiana sylvestris

Using cell suspension cultures of Nicotiana sylvestris, spontaneous oligomycin- or chloramphenicol-resistant variants were isolated at a frequency of 10⁻⁷. Treatments with ethyl methane sulfonate (EMS), a mutagen, increased this frequency 5–50 times. After 18–24 months of culture on basal medium, 6 variants retained a low level of resistance to oligomycin, 9 a high level. Four chloramphenicol-resistant variants were still slightly resistant and one was three times more resistant than the wild-type.     

Importance of ROS and antioxidant system during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation

The present study suggests the importance of reactive oxygen species (ROS) and antioxidant metabolites as biochemical signals during the beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation at saturating light and optimal CO₂. Changes in steady-state photosynthesis of pea mesophyll protoplasts monitored in the presence of antimycin A [AA, inhibitor of cytochrome oxidase (COX) pathway] and salicylhydroxamic acid [SHAM, inhibitor of alternative oxidase (AOX) pathway] were correlated with total cellular ROS and its scavenging system. Along with superoxide dismutase (SOD) and catalase (CAT), responses of enzymatic components—ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and non-enzymatic redox components of ascorbate–glutathione (Asc–GSH) cycle, which play a significant role in scavenging cellular ROS, were examined in the presence of mitochondrial inhibitors. Both AA and SHAM caused marked reduction in photosynthetic carbon assimilation with concomitant rise in total cellular ROS. Restriction of electron transport through COX or AOX pathway had differential effect on ROS generating (SOD), ROS scavenging (CAT and APX) and antioxidant (Asc and GSH) regenerating (MDAR and GR) enzymes. Further, restriction of mitochondrial electron transport decreased redox ratios of both Asc and GSH. However, while decrease in redox ratio of Asc was more prominent in the presence of SHAM in light compared with dark, decrease in redox ratio of GSH was similar in both dark and light. These results suggest that the maintenance of cellular ROS at optimal levels is a prerequisite to sustain high photosynthetic rates which in turn is regulated by respiratory capacities of COX and AOX pathways.     

Alternative methods of photosynthetic carbon assimilation in marine macroalgae

Two green macroalgae, Codium decorticatum and Udotea flabellum, differ photosynthetically. Codium had high O₂-sensitive, and Udotea low O₂-insensitive, CO₂ compensation points; Codium showed a Warburg effect at seawater dissolved inorganic carbon levels and had photorespiratory CO₂ release, whereas Udotea did not. Seawater dissolved inorganic carbon levels did not saturate photosynthesis. For Codium, but not Udotea, the Warburg effect was increased by ethoxyzolamide, a carbonic anhydrase inhibitor, at high but not low pH. Isolated chloroplasts from both macroalgae showed a Warburg effect that was ethoxyzolamide-insensitive. In both macroalgae, chloroplastic and extrachloroplastic carbonic anhydrase activity was present. P-enolpyruvate carboxykinase (PEPCK) carboxylating activity in Udotea extracts was equivalent to that of ribulose bisphosphate carboxylase, and enzyme activities for C₄ acid metabolism and P-enolpyruvate regeneration were sufficient to operate a limited C₄-like system. In Udotea, malate and aspartate were early-labeled photosynthetic products that turned over within 60 seconds. Photorespiratory compounds were much less labeled in Udotea. Low dark fixation rates ruled out Crassulacean acid metabolism. A limited C₄-like system, based on PEPCK, is hypothesized to be the mechanism reducing photorespiration in Udotea. Codium showed no evidence of photosynthetic C₄ acid metabolism. Marine macroalgae, like terrestrial angiosperms, seem to have diverse photosynthetic modes.     

热带雨林三种树苗叶片光合机构对光强的适应

对生长在不同光强(自然日光的8％,25％,50％)下西双版纳热带雨林3种木本植物团花(Anthocephalus chinensis)、玉蕊(Barringtonia pendala)和藤黄(Garrcinia hanburyi)幼苗光合机构的研究表明,随着生长光强的升高,植物叶片的光饱和点、补偿点、净光合速率和非光化学淬灭系数(NPQ)升高,而表现量子效率(AQY)、有效光化学量子产量(Fv／Fm)、光化学淬灭系数(qP)下降．在抗氧化系统中,超氧化物歧化酶(SOD)、抗坏血酸过氧化酶(APX)活性随着光强的升高而升高,而过氢化物酶(CAT)活性与生长光强的变化不一致．抗坏血酸(AsA)含量随着光强的升高而急剧上升。最能反映PFD的变化．可以认为,除与叶黄素循环有关的热耗散增大之外,植物叶片抗氧化系统的加强也是响应强光的一种保护措施．     

Phytochrome B-dependent regulation of reductive phase of photosynthetic carbon assimilation

The experiments were conducted with 10-day-old seedlings of wheat (Triticum aestivum L). Phytochrome B was activated using an array of light diodes emitting light in the red spectral region (RL) and inactivated by an array of light diodes emitting far-red light (FRL). At the end of the night dark period (8 h), activity of the chloroplastic GAP-dehydrogenase complex (the sequence of reactions: 3-PGA → 1,3-PGA → 3-GAP) was 1.0?1.2 μmol of oxidized NADPH/(min g fr wt of the leaf). When the leaves of intact plants were exposed to a maximal dose of RL (20 min at 17.5 kJ/m²), enzyme activity rose by 100–120%. Longer exposure to RL (30 and 40 min) did not cause further activation. Successive exposure to RL and FRL (20 min at 3.0 kJ/m²) completely negated a stimulatory effect of RL. It was shown that as little as 5-min-long exposure to RL increased the rate of 3-GAP formation by 20–25%, and enzyme activity rose linearly when radiation dose was elevated. Determination of the lifetime of RL-activated state by its decrease in plants placed in darkness showed that decay occurred with τ_1/2 of 50?60 min when RL was switched off. Thus, a phytochrome B-induced regulation of reducing enzyme complex governing the reductive pentose phosphate cycle was discovered. Judging from the kinetics of attenuation of the activated state, phytochrome B apparently does not affect de novo synthesis of the enzyme. Since the investigated metabolic process consists of two coupled reactions controlled by kinase and dehydrogenase, the place and mechanism of action of the phytochrome system remain unknown.     

The chloroplast genome of Nicotiana sylvestris and Nicotiana tomentosiformis: complete sequencing confirms that the Nicotiana sylvestris progenitor is the maternal genome donor of Nicotiana tabacum

The tobacco cultivar Nicotiana tabacum is a natural amphidiploid that is thought to be derived from ancestors of Nicotiana sylvestris and Nicotiana tomentosiformis. To compare these chloroplast genomes, DNA was prepared from isolated chloroplasts from green leaves of N. sylvestris and N. tomentosiformis, and subjected to whole-genome shotgun sequencing. The N. sylvestris chloroplast genome comprises of 155,941 bp and shows identical gene organization with that of N. tabacum, except one ORF. Detailed comparison revealed only seven different sites between N. tabacum and N. sylvestris; three in introns, two in spacer regions and two in coding regions. The chloroplast DNA of N. tomentosiformis is 155,745 bp long and possesses also identical gene organization with that of N. tabacum, except four ORFs and one pseudogene. However, 1,194 sites differ between these two species. Compared with N. tabacum, the nucleotide substitution in the inverted repeat was much lower than that in the single-copy region. The present work confirms that the chloroplast genome from N. tabacum was derived from an ancestor of N. sylvestris, and suggests that the rate of nucleotide substitution of the chloroplast genomes from N. tabacum and N. sylvestris is very low. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Response of photosynthetic carbon assimilation in mesophyll protoplasts to restriction on mitochondrial oxidative metabolism: Metabolites related to the redox status and sucrose biosynthesis

The patterns of cellular metabolites related to redox status and sucrose biosynthesis in mesophyll protoplasts of pea (Pisum sativum L.) were examined in the absence or presence of oligomycin (inhibitor of oxidative phosphorylation) or antimycin A (inhibitor of cytochrome pathway) or salicylhydroxamic acid (SHAM) (inhibitor of alternative pathway). The increase on illumination in the rate of photosynthesis or cellular metabolites was more at optimal CO₂ (1.0 mM NaHCO₃) compared to that at limiting CO₂ (0.1 mM NaHCO₃). Furthermore, the inhibition of photosynthesis in presence of mitochondrial inhibitors was more pronounced at optimal CO₂ than that at limiting CO₂. There was a marked increase in steady-state levels of triose-P/PGA (phosphoglyceric acid) and glucose-6-phosphate (Glc-6-P) in the presence of oligomycin and antimycin A. In contrast, SHAM caused a marked increase in malate/OAA (oxaloacetate). We suggest that dissipation of excess redox equivalents generated in photosynthesis occurs through both cytochrome and alternative pathways, while sucrose biosynthesis is backed up by cytochrome pathway alone. Thus, mitochondrial respiration (through both cytochrome and alternative pathways of mitochondrial electron transport) optimizes chloroplast photosynthesis by modulating cellular metabolites related to both intracellular redox state and sucrose biosynthesis.     

The mitochondrial external NADPH dehydrogenase modulates the leaf NADPH/NADP+ ratio in transgenic Nicotiana sylvestris

Plant mitochondria contain alternative external NAD(P)H dehydrogenases,which oxidize cytosolic NADH or NADPH and reduce ubiquinonewithout inherent linkage to proton pumping and ATP production.In potato, St-NDB1 is an external Ca²⁺-dependent NADPH dehydrogenase.The physiological function of this enzyme was investigated inhomozygous Nicotiana sylvestris lines overexpressing St-ndb1and co-suppressing St-ndb1 and an N. sylvestris ndb1. In leafmitochondria isolated from the overexpressor lines, higher activityof alternative oxidase (AOX) was detected. However, the AOXinduction was substantially weaker than in the complex I-deficientCMSII mutant, previously shown to contain elevated amounts ofNAD(P)H dehydrogenases and AOX. An aox1b and an aox2 gene wereup-regulated in CMSII, but only aox1b showed a response, albeitsmaller, in the transgenic lines, indicating differences inAOX activation between the genotypes. As in CMSII, the increaseof AOX in the overexpressing lines was not due to a generaloxidative stress. The lines overexpressing St-ndb1 had consistentlylowered leaf NADPH/NADP⁺ ratios in the light and variably decreasedlevels in darkness, but unchanged NADH/NAD⁺ ratios. CMSII insteadhad similar NADPH/NADP⁺ and lower NADH/NAD⁺ ratios than thewild type. These results demonstrate that St-NDB1 is able tomodulate the cellular balance of NADPH and NADP⁺ at least inthe day and that reduction of NADP(H) and NAD(H) is independentlycontrolled. Similar growth rates, chloroplast malate dehydrogenaseactivation and xanthophyll ratios indicate that the change inreduction does not communicate to the chloroplast, and thatthe cell tolerates significant changes in NADP(H) reductionwithout deleterious effects.