Christelle Dutilleul

Functional Mitochondrial Complex I Is Required by Tobacco Leaves for Optimal Photosynthetic Performance in Photorespiratory Conditions and during Transients

The importance of the mitochondrial electron transport chain in photosynthesis was studied using the tobacco (Nicotiana sylvestris) mutant CMSII, which lacks functional complex I. Rubisco activities and oxygen evolution at saturating CO2showed that photosynthetic capacity in the mutant was at least as high as in wild-type (WT) leaves. Despite this, steady-state photosynthesis in the mutant was reduced by 20% to 30% at atmospheric CO2 levels. The inhibition of photosynthesis was alleviated by high CO2 or low O2. The mutant showed a prolonged induction of photosynthesis, which was exacerbated in conditions favoring photorespiration and which was accompanied by increased extractable NADP-malate dehydrogenase activity. Feeding experiments with leaf discs demonstrated that CMSII had a lower capacity than the WT for glycine (Gly) oxidation in the dark. Analysis of the postillumination burst in CO2 evolution showed that this was not because of insufficient Gly decarboxylase capacity. Despite the lower rate of Gly metabolism in CMSII leaves in the dark, the Gly to Ser ratio in the light displayed a similar dependence on photosynthesis to the WT. It is concluded that: (a) Mitochondrial complex I is required for optimal photosynthetic performance, despite the operation of alternative dehydrogenases in CMSII; and (b) complex I is necessary to avoid redox disruption of photosynthesis in conditions where leaf mitochondria must oxidize both respiratory and photorespiratory substrates simultaneously.

Mitochondria-Driven Changes in Leaf NAD Status Exert a Crucial Influence on the Control of Nitrate Assimilation and the Integration of Carbon and Nitrogen Metabolism

The Nicotiana sylvestris mutant, CMS, lacks the mitochondrial gene nad7 and functional complex I, and respires using low-affinity NADH (alternative) mitochondrial dehydrogenases. Here, we show that this adjustment of respiratory pathways is associated with a profound modification of foliar carbon-nitrogen balance. CMS leaves are characterized by abundant amino acids compared to either wild-type plants or CMS in which complex I function has been restored by nuclear transformation with the nad7 cDNA. The metabolite profile of CMS leaves is enriched in amino acids with low carbon/nitrogen and depleted in starch and 2-oxoglutarate. Deficiency in 2-oxoglutarate occurred despite increased citrate and malate and higher capacity of key anaplerotic enzymes, notably the mitochondrial NAD-dependent isocitrate dehydrogenase. The accumulation of nitrogen-rich amino acids was not accompanied by increased expression of enzymes involved in nitrogen assimilation. Partitioning of 15N-nitrate into soluble amines was enhanced in CMS leaf discs compared to wild-type discs, especially in the dark. Analysis of pyridine nucleotides showed that both NAD and NADH were increased by 2-fold in CMS leaves. The growth retardation of CMS relative to the wild type was highly dependent on photoperiod, but at all photoperiod regimes the link between high contents of amino acids and NADH was observed. Together, the data provide strong evidence that (1) NADH availability is a critical factor in influencing the rate of nitrate assimilation and that (2) NAD status plays a crucial role in coordinating ammonia assimilation with the anaplerotic production of carbon skeletons.

Redox regulation of peroxiredoxin and proteinases by ascorbate and thiols during pea root nodule senescence

Redox factors contributing to nodule senescence were studied in pea. The abundance of the nodule cytosolic peroxiredoxin but not the mitochondrial peroxiredoxin protein was modulated by ascorbate. In contrast to redox‐active antioxidants such as ascorbate and cytosolic peroxiredoxin that decreased during nodule development, maximal extractable nodule proteinase activity increased progressively as the nodules aged. Cathepsin‐like activities were constant throughout development but serine and cysteine proteinase activities increased during senescence. Senescence‐induced cysteine proteinase activity was inhibited by cysteine, dithiotreitol, or E‐64. Senescence‐dependent decreases in redox‐active factors, particularly ascorbate and peroxiredoxin favour decreased redox‐mediated inactivation of cysteine proteinases.

Mitochondrial respiratory pathways modulate nitrate sensing and nitrogen-dependent regulation of plant architecture in Nicotiana sylvestris.

Mitochondrial electron transport pathways exert effects on carbon–nitrogen (C/N) relationships. To examine whether mitochondria–N interactions also influence plant growth and development, we explored the responses of roots and shoots to external N supply in wild-type (WT) Nicotiana sylvestris and the cytoplasmic male sterile II (CMSII) mutant, which has a N-rich phenotype. Root architecture in N. sylvestris seedlings showed classic responses to nitrate and sucrose availability. In contrast, CMSII showed an altered ‘nitrate-sensing’ phenotype with decreased sensitivity to C and N metabolites. The WT growth phenotype was restored in CMSII seedling roots by high nitrate plus sugars and in shoots by gibberellic acid (GA). Genome-wide cDNA-amplified fragment length polymorphism (AFLP) analysis of leaves from mature plants revealed that only a small subset of transcripts was altered in CMSII. Tissue abscisic acid content was similar in CMSII and WT roots and shoots, and growth responses to zeatin were comparable. However, the abundance of key transcripts associated with GA synthesis was modified both by the availability of N and by the CMSII mutation. The CMSII mutant maintained a much higher shoot/root ratio at low N than WT, whereas no difference was observed at high N. Shoot/root ratios were strikingly correlated with root amines/nitrate ratios, values of <1 being characteristic of high N status. We propose a model in which the amine/nitrate ratio interacts with GA signalling and respiratory pathways to regulate the partitioning of biomass between shoots and roots.

Arabidopsis Putative Selenium-Binding Protein1 Expression Is Tightly Linked to Cellular Sulfur Demand and Can Reduce Sensitivity to Stresses Requiring Glutathione for Tolerance

Selenium-Binding Protein1 (SBP1) gene expression was studied in Arabidopsis (Arabidopsis thaliana) seedlings challenged with several stresses, including cadmium (Cd), selenium {selenate [Se(VI)] and selenite [Se(IV)]}, copper (Cu), zinc (Zn), and hydrogen peroxide (H2O2) using transgenic lines expressing the luciferase (LUC) reporter gene under the control of the SBP1 promoter. In roots and shoots of SBP1∷LUC lines, LUC activity increased in response to Cd, Se(VI), Cu, and H2O2 but not in response to Se(IV) or Zn. The pattern of expression of SBP1 was similar to that of PRH43, which encodes the 5′-Adenylylphosphosulfate Reductase2, a marker for the induction of the sulfur assimilation pathway, suggesting that an enhanced sulfur demand triggers SBP1 up-regulation. Correlated to these results, SBP1 promoter showed enhanced activity in response to sulfur starvation. The sulfur starvation induction of SBP1 was abolished by feeding the plants with glutathione (GSH) and was enhanced when seedlings were treated simultaneously with buthionine sulfoxide, which inhibits GSH synthesis, indicating that GSH level participates in the regulation of SBP1 expression. Changes in total GSH level were observed in seedlings challenged with Cd, Se(VI), and H2O2. Accordingly, cad2-1 seedlings, affected in GSH synthesis, were more sensitive than wild-type plants to these three stresses. Moreover, wild-type and cad2-1 seedlings overexpressing SBP1 showed a significant enhanced tolerance to Se(VI) and H2O2 in addition to the previously described resistance to Cd, highlighting that SBP1 expression decreases sensitivity to stress requiring GSH for tolerance. These results are discussed with regard to the potential regulation and function of SBP1 in plants.

The Arabidopsis Putative Selenium-Binding Protein Family: Expression Study and Characterization of SBP1 as a Potential New Player in Cadmium Detoxification Processes

In Arabidopsis (Arabidopsis thaliana), the putative selenium-binding protein (SBP) gene family is composed of three members (SBP1–SBP3). Reverse transcription-polymerase chain reaction analyses showed that SBP1 expression was ubiquitous. SBP2 was expressed at a lower level in flowers and roots, whereas SBP3 transcripts were only detected in young seedling tissues. In cadmium (Cd)-treated seedlings, SBP1 level of expression was rapidly increased in roots. In shoots, SBP1 transcripts accumulated later and for higher Cd doses. SBP2 and SBP3 expression showed delayed or no responsiveness to Cd. In addition, luciferase (LUC) activity recorded on Arabidopsis lines expressing the LUC gene under the control of the SBP1 promoter further showed dynamic regulation of SBP1 expression during development and in response to Cd stress. Western-blot analysis using polyclonal antibodies raised against SBP1 showed that SBP1 protein accumulated in Cd-exposed tissues in correlation with SBP1 transcript amount. The sbp1 null mutant displayed no visible phenotype under normal and stress conditions that was explained by the up-regulation of SBP2 expression. SBP1 overexpression enhanced Cd accumulation in roots and reduced sensitivity to Cd in wild type and, more significantly, in Cd-hypersensitive cad mutants that lack phytochelatins. Similarly, in Saccharomyces cerevisiae, SBP1 expression led to increased Cd tolerance of the Cd-hypersensitive ycf1 mutant. In vitro experiments showed that SBP1 has the ability to bind Cd. These data highlight the importance of maintaining the adequate SBP protein level under healthy and stress conditions and suggest that, during Cd stress, SBP1 accumulation efficiently helps to detoxify Cd potentially through direct binding.

Evidence for ACD5 ceramide kinase activity involvement in Arabidopsis response to cold stress

Although sphingolipids emerged as important signals for plant response to low temperature, investigations have been limited so far to the function of long-chain base intermediates. The formation and function of ceramide phosphates (Cer-Ps) in chilled Arabidopsis were explored. Cer-Ps were analysed by thin layer chromatography (TLC) following in vivo metabolic radiolabelling. Ceramide kinase activity, gene expression and growth phenotype were determined in unstressed and cold-stressed wild type (WT) and Arabidopsis ceramide kinase mutant acd5. A rapid and transient formation of Cer-P occurs in cold-stressed WT Arabidopsis plantlets and cultured cells, which is strongly impaired in acd5 mutant. Although concomitant, Cer-P formation is independent of long-chain base phosphate (LCB-P) formation. No variation of ceramide kinase activity was measured in vitro in WT plantlets upon cold stress but the activity in acd5 mutant was further reduced by cold stress. At the seedling stage, acd5 response to cold was similar to that of WT. Nevertheless, acd5 seed germination was hypersensitive to cold and abscisic acid (ABA), and ABA-dependent gene expression was modified in acd5 seeds when germinated at low temperature. Our data involve for the first time Cer-P and ACD5 in low temperature response and further underline the complexity of sphingolipid signalling operating during cold stress.