Pyrroline-5-carboxylate dehydrogenase is an essential enzyme for proline dehydrogenase function during dark-induced senescence in Arabidopsis thaliana.

During leaf senescence, nitrogen is remobilized and carbon backbones are replenished by amino acid catabolism, with many of the key reactions occurring in mitochondria. The intermediate Δ1 -pyrroline-5-carboxylate (P5C) is common to some catabolic pathways, thus linking the metabolism of several amino acids, including proline and arginine. Specifically, mitochondrial proline catabolism involves sequential action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to produce P5C and then glutamate. Arginine catabolism produces urea and ornithine, the latter in the presence of α-ketoglutarate being converted by ornithine δ-aminotransferase (OAT) into P5C and glutamate. Metabolic changes during dark-induced leaf senescence (DIS) were studied in Arabidopsis thaliana leaves of Col-0 and in prodh1prodh2, p5cdh and oat mutants. Progression of DIS was followed by measuring chlorophyll and proline contents for 5 days. Metabolomic profiling of 116 compounds revealed similar profiles of Col-0 and oat metabolism, distinct from prodh1prodh2 and p5cdh metabolism. Metabolic dynamics were accelerated in p5cdh by 1 day. Notably, more P5C and proline accumulated in p5cdh than in prodh1prodh2. ProDH1 enzymatic activity and protein amount were significantly down-regulated in p5cdh mutant at Day 4 of DIS. Mitochondrial P5C levels appeared critical in determining the flow through interconnected amino acid remobilization pathways to sustain senescence.

Proline oxidation fuels mitochondrial respiration during dark-induced leaf senescence in Arabidopsis thaliana.

Leaf senescence is a form of developmentally programmed cell death that allows the remobilization of nutrients and cellular materials from leaves to sink tissues and organs. Among the catabolic reactions that occur upon senescence, little is known about the role of proline catabolism. In this study, the involvement in dark-induced senescence of proline dehydrogenases (ProDHs), which catalyse the first and rate-limiting step of proline oxidation in mitochondria, was investigated using prodh single- and double-mutants with the help of biochemical, proteomic, and metabolomic approaches. The presence of ProDH2 in mitochondria was confirmed by mass spectrometry and immunogold labelling in dark-induced leaves of Arabidopsis. The prodh1 prodh2 mutant exhibited enhanced levels of most tricarboxylic acid cycle intermediates and free amino acids, demonstrating a role of ProDH in mitochondrial metabolism. We also found evidence of the involvement and the importance of ProDH in respiration, with proline as an alternative substrate, and in remobilization of proline during senescence to generate glutamate and energy that can then be exported to sink tissues and organs.

Phospholipases Dζ1 and Dζ2 have distinct roles in growth and antioxidant systems in Arabidopsis thaliana responding to salt stress.

MAIN CONCLUSION: Phospholipases Dζ play different roles in Arabidopsis salt tolerance affecting the regulation of ion transport and antioxidant responses. Lipid signalling mediated by phospholipase D (PLD) plays essential roles in plant growth including stress and hormonal responses. Here we show that PLDζ1 and PLDζ2 have distinct effects on Arabidopsis responses to salinity. A transcriptome analysis of a double pldζ1pldζ2 mutant revealed a cluster of genes involved in abiotic and biotic stresses, such as the high salt-stress responsive genes DDF1 and RD29A. Another cluster of genes with a common expression pattern included ROS detoxification genes involved in electron transport and biotic and abiotic stress responses. Total superoxide dismutase (SOD) activity was induced early in the shoots and roots of all pldζ mutants exposed to mild or severe salinity with the highest SOD activity measured in pldζ2 at 14 days. Lipid peroxidation in shoots and roots was higher in the pldζ1 mutant upon salt treatment and pldζ1 accumulated H2O2 earlier than other genotypes in response to salt. Salinity caused less deleterious effects on K+ accumulation in shoots and roots of the pldζ2 mutant than of wild type, causing only a slight variation in Na+/K+ ratio. Relative growth rates of wild-type plants, pldζ1, pldζ2 and pldζ1pldζ2 mutants were similar in control conditions, but strongly affected by salt in WT and pldζ1. The efficiency of photosystem II, estimated by measuring the ratio of chlorophyll fluorescence (F v/F m ratio), was strongly decreased in pldζ1 under salt stress. In conclusion, PLDζ2 plays a key role in determining Arabidopsis sensitivity to salt stress allowing ion transport and antioxidant responses to be finely regulated.

Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana.

Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140 kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain.

Arabidopsis A BOUT DE SOUFFLE is a putative mitochondrial transporter involved in photorespiratory metabolism and is required for meristem growth at ambient CO2 levels.

Photorespiratory metabolism is essential in all oxygenic photosynthetic organisms. In plants, it is a highly compartmentalized pathway that involves chloroplasts, peroxisomes, mitochondria and the cytoplasm. The metabolic pathway itself is well characterized, and the enzymes required for its function have been identified. However, very little information is available on the transport proteins that catalyze the high metabolic flux between the involved compartments. Here we show that the A BOUT DE SOUFFLE (BOU) gene, which encodes a mitochondrial carrier, is involved in photorespiration in Arabidopsis. BOU was found to be co-expressed with photorespiratory genes in leaf tissues. The knockout mutant bou-2 showed the hallmarks of a photorespiratory growth phenotype, an elevated CO(2) compensation point, and excessive accumulation of glycine. Furthermore, degradation of the P-protein, a subunit of glycine decarboxylase, was demonstrated for bou-2, and is reflected in strongly reduced glycine decarboxylase activity. The photorespiration defect in bou-2 has dramatic consequences early in the seedling stage, which are highlighted by transcriptome studies. In bou-2 seedlings, as in shm1, another photorespiratory mutant, the shoot apical meristem organization is severely compromised. Cell divisions are arrested, leading to growth arrest at ambient CO(2) . Although the specific substrate for the BOU transporter protein remains elusive, we show that it is essential for the function of the photorespiratory metabolism. We hypothesize that BOU function is linked with glycine decarboxylase activity, and is required for normal apical meristems functioning in seedlings.

Mutations in the hyperosmotic stress-responsive mitochondrial BASIC AMINO ACID CARRIER2 enhance proline accumulation in Arabidopsis.

Mitochondrial carrier family proteins are diverse in their substrate specificity, organellar location, and gene expression. In Arabidopsis (Arabidopsis thaliana), 58 genes encode these six-transmembrane-domain proteins. We investigated the biological role of the basic amino acid carrier Basic Amino Acid Carrier2 (BAC2) from Arabidopsis that is structurally and functionally similar to ARG11, a yeast ornithine and arginine carrier, and to Arabidopsis BAC1. By studying the expression of BAC2 and the consequences of its mutation in Arabidopsis, we showed that BAC2 is a genuine mitochondrial protein and that Arabidopsis requires expression of the BAC2 gene in order to use arginine. The BAC2 gene is induced by hyperosmotic stress (with either 0.2 m NaCl or 0.4 m mannitol) and dark-induced senescence. The BAC2 promoter contains numerous stress-related cis-regulatory elements, and the transcriptional activity of BAC2:beta-glucuronidase is up-regulated by stress and senescence. Under hyperosmotic stress, bac2 mutants express the P5CS1 proline biosynthetic gene more strongly than the wild type, and this correlates with a greater accumulation of Pro. Our data suggest that BAC2 is a hyperosmotic stress-inducible transporter of basic amino acids that contributes to proline accumulation in response to hyperosmotic stress in Arabidopsis.

Variation in relative water content, proline accumulation and stress gene expression in two cowpea landraces under drought.

Many landraces of cowpea [Vigna unguiculata (L.) Walp.] are adapted to particular geographical and climatic conditions. Here we describe two landraces grown respectively in arid and temperate areas of Algeria and assess their physiological and molecular responses to drought stress. As expected, when deprived of water cowpea plants lose water over time with a gradual reduction in transpiration rate. The landraces differed in their relative water content (RWC) and whole plant transpiration rate. The landrace from Menia, an arid area, retained more water in adult leaves. Both landraces responded to drought stress at the molecular level by increasing expression of stress-related genes in aerial parts, including proline metabolism genes. Expression of gene(s) encoding proline synthesis enzyme P5CS was up regulated and gene expression of ProDH, a proline catabolism enzyme, was down regulated. Relatively low amounts of proline accumulated in adult leaves with slight differences between the two landraces. During drought stress the most apical part of plants stayed relatively turgid with a high RWC compared to distal parts that wilted. Expression of key stress genes was higher and more proline accumulated at the apex than in distal leaves indicating that cowpea has a non-uniform stress response at the whole plant level. Our study reveals a developmental control of water stress through preferential proline accumulation in the upper tier of the cowpea plant. We also conclude that cowpea landraces display physiological adaptations to water stress suited to the arid and temperate climates in which they are cultivated.

Phaseolus vulgaris L. Seedlings Exposed to Prometryn Herbicide Contaminated Soil Trigger an Oxidative Stress Response.

Herbicides from the family of S-triazines, such as prometryn, have been widely used in crop production and can constitute an environmental pollution in both water and soil. As a valuable crop, the common bean (Phaseolus vulgaris L.) is grown all over the world and could be exposed to such herbicides. We wanted to investigate the possible stress sustained by the common bean growing in prometryn-polluted soil. Two situations were observed: when soil was treated with ≥100 µM prometryn, some, but not all, measured growth parameters were affected in a dose-dependent manner. Growth was reduced, and photosynthetic pigments and photosynthetic products were less accumulated when soil was treated with ≥100 µM prometryn. Reactive oxygen species (ROS) produced had a deleterious effect, as seen by the accumulation of oxidized lipid in the form of malondialdehyde (MDA). Higher prometryn (500 µM) concentrations had a disastrous effect, reducing antioxidant activities. At a low (10 µM) concentration, prometryn increased antioxidant enzymatic activities without affecting plant growth or MDA production. Gene expression of proline metabolism genes and proline accumulation confirm that bean plants respond to a stress according to the prometryn concentration. Physiological responses such as antioxidative enzymes APX, CAT, and the enzyme implicated in the metabolization of xenobiotics, GST, were increased at 10 and 100 µM, which indicated a prevention of deleterious effects of prometryn, suggesting that bean is a suitable material both for herbicide pollution sensing and as a crop on a low level of herbicide pollution.

BASIC AMINO ACID CARRIER 2 gene expression modulates arginine and urea content and stress recovery in Arabidopsis leaves.

In plants, basic amino acids are important for the synthesis of proteins and signaling molecules and for nitrogen recycling. The Arabidopsis nuclear gene BASIC AMINO ACID CARRIER 2 (BAC2) encodes a mitochondria-located carrier that transports basic amino acids in vitro. We present here an analysis of the physiological and genetic function of BAC2 in planta. When BAC2 is overexpressed in vivo, it triggers catabolism of arginine, a basic amino acid, leading to arginine depletion and urea accumulation in leaves. BAC2 expression was known to be strongly induced by stress. We found that compared to wild type plants, bac2 null mutants (bac2-1) recover poorly from hyperosmotic stress when restarting leaf expansion. The bac2-1 transcriptome differs from the wild-type transcriptome in control conditions and under hyperosmotic stress. The expression of genes encoding stress-related transcription factors (TF), arginine metabolism enzymes, and transporters is particularly disturbed in bac2-1, and in control conditions, the bac2-1 transcriptome has some hallmarks of a wild-type stress transcriptome. The BAC2 carrier is therefore involved in controlling the balance of arginine and arginine-derived metabolites and its associated amino acid metabolism is physiologically important in equipping plants to respond to and recover from stress.

Arabidopsis A BOUT DE SOUFFLE, which is homologous with mammalian carnitine acyl carrier, is required for postembryonic growth in the light.

The degradation of storage compounds just after germination is essential to plant development, providing energy and molecules necessary for the building of a photosynthetic apparatus and allowing autotrophic growth. We identified à bout de souffle (bou), a new Arabidopsis mutation. Mutant plants stopped developing after germination and degraded storage lipids, but they did not proceed to autotrophic growth. Neither leaves nor roots developed in the mutant. However, externally added sugar or germination in the dark could bypass this developmental block and allowed mutant plants to develop. The mutated gene was cloned using the transposon Dissociation as a molecular tag. The gene coding sequence showed similarity to those of the mitochondrial carnitine acyl carriers (CACs) or CAC-like proteins. In animals and yeast, these transmembrane proteins are involved in the transport of lipid-derived molecules across mitochondrial membranes for energy and carbon supply. The data presented here suggest that BOU identifies a novel mitochondrial pathway that is necessary to seedling development in the light. The BOU pathway would be an alternative to the well-known glyoxylate pathway.

Molecular and physiological characterization of arabidopsis GAI alleles obtained in targeted Ds-tagging experiments.

Bioactive gibberellin (GA) is an essential regulator of vascular plant development. The GAI gene of Arabidopsis thaliana (L.) Heynh. encodes a product (GAI) that is involved in GA signalling. The dominant mutant gai allele encodes an altered product (gai) that confers reduced GA responses, dwarfism, and elevated endogenous GA levels. Recessive, presumed loss-of-function alleles of GAI confer normal height and resistance to the GA biosynthesis inhibitor paclobutrazol. One explanation for these observations is that GAI is a growth repressor whose activity is opposed by GA, whilst gai retains a constitutive repressor activity that is less affected by GA. Previously, we described gai-t6, a mutant allele which contains an insertion of a maize Ds transposable element into gai. Here we describe the molecular and physiological characterization of two further alleles (gai-t5, gai-t7) identified during the Ds mutagenesis experiment. These alleles confer paclobutrazol resistance and normal endogenous GA levels. Thus the phenotype conferred by gai-t5, gai-t6 and gai-t7 is not due to elevated GA levels, but is due to loss of gai, a constitutively active plant growth repressor.

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation.

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5'-end and the long 3'-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.