The Arabidopsis DUF239 gene family encodes Neprosin-like proteins that are widely expressed in seed endosperm.

Domain of unknown function 239 (DUF239) is a conserved sequence found in the catalytic site of Neprosins which are specific secreted prolyl endopeptidases found in the Nepenthes genus. Neprosins participate in the nitrogen cycle by digesting preys trapped in the pitcher of these carnivorous plants. Apart from that, DUF239s have been poorly documented in plants. We have identified 50 genes containing DUF239-coding sequences in the Arabidopsis genome that are distributed across six distinct phylogenetic clusters. The chromosomal distribution suggests that several genes are the result of recent duplication events, with up to eight genes found in a strict tandem distribution. In Arabidopsis, most of DUF239-containing sequences are also associated to a Neprosin-activating domain (DUF4409) and an amino-terminal α-helix which corresponds to the typical domain organization of the Neprosins described in the Nepenthes genus. Analysis of Arabidopsis transcriptomic datasets reveals that 39 genes are exclusively expressed in reproductive organs, mainly during seed development and more specifically in the endosperm (23 genes). The peculiar expression pattern of the DUF239 gene family in Arabidopsis suggests new functions of Neprosin-like proteins in plants during seed development.

Tagging and Capture of Prenylated CaaX-Proteins from Plant Cell Cultures.

The tagging-via-substrate strategy allows the probing of in vivo post-translationally modified proteins thanks to a labeled substrate. This method has been used for the detection and proteomic analysis of prenylated proteins in mammals and more recently in plants. It consists of the labeling of prenylated proteins by supplying azido-prenyl to cells. The azido-prenylated proteins are then selectively linked to biotin alkyne, which allows their capture using streptavidin beads, and their subsequent identification by mass spectrometry. In this chapter, we describe this procedure on Arabidopsis cell suspension and how it can be applied for Catharanthus roseus cells.

Protein Farnesylation Takes Part in Arabidopsis Seed Development.

Protein farnesylation is a post-translational modification regulated by the ERA1 (Enhanced Response to ABA 1) gene encoding the ß-subunit of the protein farnesyltransferase in Arabidopsis. The era1 mutants have been described for over two decades and exhibit severe pleiotropic phenotypes, affecting vegetative and flower development. We further investigated the development and quality of era1 seeds. While the era1 ovary contains numerous ovules, the plant produces fewer seeds but larger and heavier, with higher protein contents and a modified fatty acid distribution. Furthermore, era1 pollen grains show lower germination rates and, at flower opening, the pistils are immature and the ovules require one additional day to complete the embryo sac. Hand pollinated flowers confirmed that pollination is a major obstacle to era1 seed phenotypes, and a near wild-type seed morphology was thus restored. Still, era1 seeds conserved peculiar storage protein contents and altered fatty acid distributions. The multiplicity of era1 phenotypes reflects the diversity of proteins targeted by the farnesyltransferase. Our work highlights the involvement of protein farnesylation in seed development and in the control of traits of agronomic interest.

Remarkable Evolutionary Conservation of Antiobesity ADIPOSE/WDTC1 Homologs in Animals and Plants.

ASG2 (Altered Seed Germination 2) is a prenylated protein in Arabidopsis thaliana that participates to abscisic acid signaling and is proposed to act as a substrate adaptor for the DDB1 (DNA damage-binding protein 1)-CUL4 (Cullin 4) E3 ubiquitin ligase complex. ASG2 harbors WD40 and TetratricoPeptide Repeat (TPR) domains, and resembles the well-conserved animal gene called ADP (antiobesity factor ADIPOSE) in fly and WDTC1 (WD40 and TPR 1) in humans. Loss of function of WDTC1 results in an increase in adipocytes, fat accumulation, and obesity. Antiadipogenic functions of WDTC1 involve regulation of fat-related gene transcription, notably through its binding to histone deacetylases (HDACs). Our sequence and phylogenetic analysis reveals that ASG2 belongs to the ADP/WDTC1 cluster. ASG2 and WDTC1 share a highly conserved organization that encompasses structural and functional motifs: seven WD40 domains and WD40 hotspot-related residues, three TPR protein-protein interaction domains, DDB1-binding elements [H-box and DWD (DDB1-binding WD40 protein)-box], and a prenylatable C-terminus. Furthermore, ASG2 involvement in fat metabolism was confirmed by reverse genetic approaches using asg2 knockout Arabidopsis plants. Under limited irradiance, asg2 mutants produce "obese" seeds characterized by increased weight, oil body density, and higher fatty acid contents. In addition, considering some ASG2- and WDTC1-peculiar properties, we show that the WDTC1 C-terminus is prenylated in vitro and HDAC-binding capability is conserved in ASG2, suggesting that the regulation mechanism and targets of ADP/WDTC1-like proteins may be conserved features. Our findings reveal the remarkable evolutionary conservation of the structure and the physiological role of ADIPOSE homologs in animals and plants.

ASG2 is a farnesylated DWD protein that acts as ABA negative regulator in Arabidopsis.

The tagging-via-substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide-modified farnesyl moiety and captured thanks to biotin alkyne Click-iT® chemistry with further streptavidin-affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C-terminal CaaX-box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.

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.

Role of protein farnesylation events in the ABA-mediated regulation of the Pinoresinol-Lariciresinol Reductase 1 (LuPLR1) gene expression and lignan biosynthesis in flax (Linum usitatissimum L.).

A Linum usitatissimum LuERA1 gene encoding a putative ortholog of the ERA1 (Enhanced Response to ABA 1) gene of Arabidopsis thaliana (encoding the beta subunit of a farnesyltransferase) was analyzed in silico and for its expression in flax. The gene and the protein sequences are highly similar to other sequences already characterized in plants and all the features of a farnesyltransferase were detected. Molecular modeling of LuERA1 protein confirmed its farnesyltransferase nature. LuERA1 is expressed in the vegetative organs and also in the outer seedcoat of the flaxseed, where it could modulate the previously observed regulation operated by ABA on lignan synthesis. This effect could be mediated by the regulation of the transcription of a key gene for lignan synthesis in flax, the LuPLR1 gene, encoding a pinoresinol lariciresinol reductase. The positive effect of manumycin A, a specific inhibitor of farnesyltransferase, on lignan biosynthesis in flax cell suspension systems supports the hypothesis of the involvement of such an enzyme in the negative regulation of ABA action. In Arabidopsis, ERA1 is able to negatively regulate the ABA effects and the mutant era1 has an enhanced sensitivity to ABA. When expressed in an Arabidopsis cell suspension (heterologous system) LuERA1 is able to reverse the effect of the era1 mutation. RNAi experiments in flax targeting the farnesyltransferase ß-subunit encoded by the LuERA1 gene led to an increase LuPLR1 expression level associated with an increased content of lignan in transgenic calli. Altogether these results strongly suggest a role of the product of this LuERA1 gene in the ABA-mediated upregulation of lignan biosynthesis in flax cells through the activation of LuPLR1 promoter. This ABA signaling pathway involving ERA1 probably acts through the ABRE box found in the promoter sequence of LuPLR1, a key gene for lignan synthesis in flax, as demonstrated by LuPLR1 gene promoter-reporter experiments in flax cells using wild type and mutated promoter sequences.

Efficient gene targeting in a Candida guilliermondii non-homologous end-joining pathway-deficient strain.

The yeast, Candida guilliermondii, has been widely studied due to its biotechnological interest as well as its biological control potential. It integrates foreign DNA predominantly via ectopic events, likely through the well-known non-homologous end-joining (NHEJ) pathway involving the Ku70p/Ku80p heterodimer, Lig4p, Nej1p and Lif1p. This phenomenon remains highly deleterious for targeted gene knock-out strategies that require the homologous recombination process. Here, we have constructed a ku70 mutant strain derived from the ATCC 6260 reference strain of C. guilliermondii. Following a series of disruption attempts of various genes (FCY1, ADE2 and TRP5), using several previously described dominant selectable markers (URA5, SAT-1 and HPH#), we demonstrated that the efficiencies of homologous gene targeting in such a NHEJ-deficient strain was very high compared to the wild type strain. The C. guilliermondii ku70 deficient mutant thus represents a powerful recipient strain to knock-out genes efficiently in this yeast.

A TRP5/5-fluoroanthranilic acid counter-selection system for gene disruption in Candida guilliermondii.

Candida guilliermondii is an interesting biotechnological model for the industrial production of value-added metabolites and also remains an opportunistic emerging fungal agent of candidiasis often associated with oncology patients. The aim of the present study was to establish a convenient transformation system for C. guilliermondii by developing both an ATCC 6260-derived recipient strain and a recyclable selection marker. We first disrupted the TRP5 gene in the wild-type strain and demonstrated that trp5 mutants were tryptophan auxotroph as well as being resistant to the antimetabolite 5-fluoroanthranilic acid (FAA). Following an FAA selection of spontaneous mutants derived from the ATCC 6260 strain and complementation analysis, we demonstrated that trp5 genotypes could be directly recovered on FAA-containing medium. The TRP5 wild-type allele, flanked by two short repeated sequences of its 3'UTR, was then used to disrupt the FCY1 gene in C. guilliermondii trp5 recipient strains. The resulting fcy1 mutants displayed strong flucytosine resistance and a counter-selection on FAA allowed us to pop-out the TRP5 allele from the FCY1 locus. To illustrate the capacity of this blaster system to achieve a second round of gene disruption, we knocked out both the LEU2 and the HOG1 genes in the trp5, fcy1 background. Although all previously described yeast "TRP blaster" disruption systems used TRP1 as counter-selectable marker, this study demonstrated the potential of the TRP5 gene in such strategies. This newly created "TRP5 blaster" disruption system thus represents a powerful genetic tool to study the function of a large pallet of genes in C. guilliermondii.

Phytosphingosine-phosphate is a signal for AtMPK6 activation and Arabidopsis response to chilling.

• Long-chain bases (LCBs) are pleiotropic sphingolipidic signals in eukaryotes. We investigated the source and function of phytosphingosine-1-phosphate (PHS-P), a phospho-LCB rapidly and transiently formed in Arabidopsis thaliana on chilling. • PHS-P was analysed by thin-layer chromatography following in vivo metabolic radiolabelling. Pharmacological and genetic approaches were used to identify the sphingosine kinase isoforms involved in cold-responsive PHS-P synthesis. Gene expression, mitogen-activated protein kinase activation and growth phenotypes of three LCB kinase mutants (lcbk1, sphk1 and lcbk2) were studied following cold exposure. • Chilling provoked the rapid and transient formation of PHS-P in Arabidopsis cultured cells and plantlets. Cold-evoked PHS-P synthesis was reduced by LCB kinase inhibitors and abolished in the LCB kinase lcbk2 mutant, but not in lcbk1 and sphk1 mutants. lcbk2 presented a constitutive AtMPK6 activation at 22°C. AtMPK6 activation was also triggered by PHS-P treatment independently of PHS/PHS-P balance. lcbk2 mutants grew comparably with wild-type plants at 22 and 4°C, but exhibited a higher root growth at 12°C, correlated with an altered expression of the cold-responsive DELLA gene RGL3. • Together, our data indicate a function for LCBK2 in planta. Furthermore, they connect PHS-P formation with plant response to cold, expanding the field of LCB signalling in plants.

The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis.

Farnesyl diphosphate (FPP) synthase (FPS: EC.2.5.1.1, EC.2.5.1.10) catalyzes the formation of FPP from isopentenyl diphosphate and dimethylallyl diphosphate via two successive condensation reactions. A cDNA designated CrFPS, encoding a protein showing high similarities with trans-type short FPS isoforms, was isolated from the Madagascar periwinkle (Catharanthus roseus). This cDNA was shown to functionally complement the lethal FPS deletion mutant in the yeast Saccharomyces cerevisiae. At the subcellular level, while short FPS isoforms are usually described as cytosolic proteins, we showed, using transient transformations of C. roseus cells with yellow fluorescent protein-fused constructs, that CrFPS is targeted to peroxisomes. This finding is discussed in relation to the subcellular distribution of FPS isoforms in plants and animals and opens new perspectives towards the understanding of isoprenoid biosynthesis.

Nitric oxide participates in cold-responsive phosphosphingolipid formation and gene expression in Arabidopsis thaliana.

Chilling triggers rapid molecular responses that permit the maintenance of plant cell homeostasis and plant adaptation. Recent data showed that nitric oxide (NO) is involved in plant acclimation and tolerance to cold. The participation of NO in the early transduction of the cold signal in Arabidopsis thaliana was investigated. The production of NO after a short exposure to cold was assessed using the NO-sensitive fluorescent probe 4, 5-diamino fluoresceine diacetate and chemiluminescence. Pharmacological and genetic approaches were used to analyze NO sources and NO-mediated changes in cold-regulated gene expression, phosphatidic acid (PtdOH) synthesis and sphingolipid phosphorylation. NO production was detected after 1-4h of chilling. It was impaired in the nia1nia2 nitrate reductase mutant. Moreover, NO accumulation was not observed in H7 plants overexpressing the A. thaliana nonsymbiotic hemoglobin Arabidopsis haemoglobin 1 (AHb1). Cold-regulated gene expression was affected in nia1nia2 and H7 plants. The synthesis of PtdOH upon chilling was not modified by NO depletion. By contrast, the formation of phytosphingosine phosphate and ceramide phosphate, two phosphorylated sphingolipids that are transiently synthesized upon chilling, was negatively regulated by NO. Taken together, these data suggest a new function for NO as an intermediate in gene regulation and lipid-based signaling during cold transduction.

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 (H(2)O(2)) using transgenic lines expressing the luciferase (LUC) reporter gene under the control of the SBP1 promoter. In roots and shoots of SBP1LUC lines, LUC activity increased in response to Cd, Se(VI), Cu, and H(2)O(2) 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 H(2)O(2). 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 H(2)O(2) 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.

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.

Genetic variation in pea (Pisum sativum L.) demonstrates the importance of root but not shoot C/N ratios in the control of plant morphology and reveals a unique relationship between shoot length and nodulation intensity.

Nodule numbers are regulated through systemic auto-regulatory signals produced by shoots and roots. The relative effects of shoot and root genotype on nodule numbers together with relationships to organ biomass, carbon (C) and nitrogen (N) status, and related parameters were measured in pea (Pisum sativum) exploiting natural genetic variation in maturity and apparent nodulation intensity. Reciprocal grafting experiments between the early (Athos), intermediate (Phönix) and late (S00182) maturity phenotypes were performed and Pearson's correlation coefficients for the parameters were calculated. No significant correlations were found between shoot C/N ratios and plant morphology parameters, but the root C/N ratio showed a strong correlation with root fresh and dry weights as well as with shoot fresh weight with less significant interactions with leaf number. Hence, the root C/N ratio rather than shoot C/N had a predominant influence on plant morphology when pea plants are grown under conditions of symbiotic nitrogen supply. The only phenotypic characteristic that showed a statistically significant correlation with nodulation intensity was shoot length, which accounted for 68.5% of the variation. A strong linear relationship was demonstrated between shoot length and nodule numbers. Hence, pea nodule numbers are controlled by factors related to shoot extension, but not by shoot or root biomass accumulation, total C or total N. The relationship between shoot length and nodule numbers persisted under field conditions. These results suggest that stem height could be used as a breeding marker for the selection of pea cultivars with high nodule numbers and high seed N contents.

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.

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 (15)N-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.

Use of mitochondrial electron transport mutants to evaluate the effects of redox state on photosynthesis, stress tolerance and the integration of carbon/nitrogen metabolism.

Primary leaf metabolism requires the co-ordinated production and use of carbon skeletons and redox equivalents in several subcellular compartments. The role of the mitochondria in leaf metabolism has long been recognized, but it is only recently that molecular tools and mutants have become available to evaluate cause-and-effect relationships. In particular, analysis of the CMSII mutant of Nicotiana sylvestris, which lacks functional complex I, has provided information on the role of mitochondrial electron transport in leaf function. The essential feature of CMSII is the absence of a major NADH sink, i.e. complex I. This necessitates re-adjustment of whole-cell redox homeostasis, gene expression, and also influences metabolic pathways that use pyridine nucleotides. In air, CMSII is not able to use its photosynthetic capacity as well as the wild type. The mutant shows up-regulation of the leaf antioxidant system, lower leaf contents of reactive oxygen species, and enhanced stress resistance. Lastly, the loss of a major mitochondrial dehydrogenase has important repercussions for the integration of primary carbon and nitrogen metabolism, causing distinct changes in leaf organic acid profiles, and also affecting downstream processes such as the biosynthesis of the spectrum of leaf amino acids.

Leaf mitochondria modulate whole cell redox homeostasis, set antioxidant capacity, and determine stress resistance through altered signaling and diurnal regulation.

To explore the role of plant mitochondria in the regulation of cellular redox homeostasis and stress resistance, we exploited a Nicotiana sylvestris mitochondrial mutant. The cytoplasmic male-sterile mutant (CMSII) is impaired in complex I function and displays enhanced nonphosphorylating rotenone-insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive (alternative oxidase) respiration. Loss of complex I function is not associated with increased oxidative stress, as shown by decreased leaf H(2)O(2) and the maintenance of glutathione and ascorbate content and redox state. However, the expression and activity of several antioxidant enzymes are modified in CMSII. In particular, diurnal patterns of alternative oxidase expression are lost, the relative importance of the different catalase isoforms is modified, and the transcripts, protein, and activity of cytosolic ascorbate peroxidase are enhanced markedly. Thus, loss of complex I function reveals effective antioxidant crosstalk and acclimation between the mitochondria and other organelles to maintain whole cell redox balance. This reorchestration of the cellular antioxidative system is associated with higher tolerance to ozone and Tobacco mosaic virus.