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.     

Involvement of a glycerol-3-phosphate dehydrogenase in modulating the NADH/NAD+ ratio provides evidence of a mitochondrial glycerol-3-phosphate shuttle in Arabidopsis

A mitochondrial glycerol-3-phosphate (G-3-P) shuttle that channels cytosolic reducing equivalent to mitochondria for respiration through oxidoreduction of G-3-P has been extensively studied in yeast and animal systems. Here, we report evidence for the operation of such a shuttle in Arabidopsis thaliana. We studied Arabidopsis mutants defective in a cytosolic G-3-P dehydrogenase, GPDHc1, which, based on models described for other systems, functions as the cytosolic component of a G-3-P shuttle. We found that the gpdhc1 T-DNA insertional mutants exhibited increased NADH/NAD+ ratios compared with wild-type plants under standard growth conditions, as well as impaired adjustment of NADH/NAD+ ratios under stress simulated by abscisic acid treatment. The altered redox state of the NAD(H) pool was correlated with shifts in the profiles of metabolites concerning intracellular redox exchange. The impairment in maintaining cellular redox homeostasis was manifest by a higher steady state level of reactive oxygen species under standard growth conditions and by a significantly augmented hydrogen peroxide production under stress. Loss of GPDHc1 affected mitochondrial respiration, particularly through a diminished capacity of the alternative oxidase respiration pathway. We propose a model that outlines potential involvements of a mitochondrial G-3-P shuttle in plant cells for redox homeostasis.     

Disruption of alternative NAD(P)H dehydrogenases leads to decreased mitochondrial ROS in Neurospora crassa

Mitochondria are a main providers of high levels of energy, but also a major source of reactive oxygen species (ROS) during normal oxidative metabolism. The involvement of Neurospora crassa alternative NAD(P)H dehydrogenases in mitochondrial ROS production was evaluated. The growth responses of a series of respiratory mutants to several stress conditions revealed that disrupting alternative dehydrogenases leads to an increased tolerance to the redox cycler paraquat, with a mutant devoid of the external NDE1 and NDE2 enzymes being significantly more resistant. The nde1nde2 mutant mitochondria show a significant decrease in ROS generation in the presence and absence of paraquat, regardless of the respiratory substrate used, and an intrinsic increase in catalase activity. Analysis of ROS production by a complex I mutant (nuo51) indicates that, as in other organisms, paraquat-derived ROS in Neurospora mitochondria occur mainly at the level of complex I. We propose that disruption of the external NAD(P)H dehydrogenases NDE1 and NDE2 leads to a synergistic effect diminishing ROS generation by the mitochondrial respiratory chain. This, in addition to a robust increase in scavenging capacity, provides the mutant strain with an improved ability to withstand paraquat treatment.     

Ca2+-binding and Ca2+-independent respiratory NADH and NADPH dehydrogenases of Arabidopsis thaliana

Type II NAD(P)H:quinone oxidoreductases are single polypeptide proteins widespread in the living world. They bypass the first site of respiratory energy conservation, constituted by the type I NADH dehydrogenases. To investigate substrate specificities and Ca(2+) binding properties of seven predicted type II NAD(P)H dehydrogenases of Arabidopsis thaliana we have produced them as T7-tagged fusion proteins in Escherichia coli. The NDB1 and NDB2 enzymes were found to bind Ca(2+), and a single amino acid substitution in the EF hand motif of NDB1 abolished the Ca(2+) binding. NDB2 and NDB4 functionally complemented an E. coli mutant deficient in endogenous type I and type II NADH dehydrogenases. This demonstrates that these two plant enzymes can substitute for the NADH dehydrogenases in the bacterial respiratory chain. Three NDB-type enzymes displayed distinct catalytic profiles with substrate specificities and Ca(2+) stimulation being considerably affected by changes in pH and substrate concentrations. Under physiologically relevant conditions, the NDB1 fusion protein acted as a Ca(2+)-dependent NADPH dehydrogenase. NDB2 and NDB4 fusion proteins were NADH-specific, and NDB2 was stimulated by Ca(2+). The observed activity profiles of the NDB-type enzymes provide a fundament for understanding the mitochondrial system for direct oxidation of cytosolic NAD(P)H in plants. Our findings also suggest different modes of regulation and metabolic roles for the analyzed A. thaliana enzymes.     

L-galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation.     

MPK6, sphinganine and the LCB2a gene from serine palmitoyltransferase are required in the signaling pathway that mediates cell death induced by long chain bases in Arabidopsis

Long chain bases (LCBs) are sphingolipid intermediates acting as second messengers in programmed cell death (PCD) in plants. Most of the molecular and cellular features of this signaling function remain unknown. We induced PCD conditions in Arabidopsis thaliana seedlings and analyzed LCB accumulation kinetics, cell ultrastructure and phenotypes in serine palmitoyltransferase (spt), mitogen-activated protein kinase (mpk), mitogen-activated protein phosphatase (mkp1) and lcb-hydroxylase (sbh) mutants. The lcb2a-1 mutant was unable to mount an effective PCD in response to fumonisin B1 (FB1), revealing that the LCB2a gene is essential for the induction of PCD. The accumulation kinetics of LCBs in wild-type (WT) and lcb2a-1 plants and reconstitution experiments with sphinganine indicated that this LCB was primarily responsible for PCD elicitation. The resistance of the null mpk6 mutant to manifest PCD on FB1 and sphinganine addition and the failure to show resistance on pathogen infection and MPK6 activation by FB1 and LCBs indicated that MPK6 mediates PCD downstream of LCBs. This work describes MPK6 as a novel transducer in the pathway leading to LCB-induced PCD in Arabidopsis, and reveals that sphinganine and the LCB2a gene are required in a PCD process that operates as one of the more effective strategies used as defense against pathogens in plants.     

A partially assembled complex I in NAD4-deficient mitochondria of maize

The proton-translocating NADH:ubiquinone oxidoreductase (respiratory complex I) consists of at least 32 subunits in higher plants, nine of which are mitochondrially encoded (NAD 1–7, NAD4L, NAD9). Complex I (CI) has been analyzed from a mitochondrial mutant of maize, NCS2, that carries a deletion for the 3′ end of the nad4 gene. Mitochondria from highly defective, near-homoplasmic mutant plants have only trace amounts of the normal complex I. Instead, a reduced amount of a smaller complex, which also exhibits NADH dehydrogenase activity, is detected on ‘blue-native’ polyacrylamide gels. Subunits of 76 kDa, 40 kDa and 55 kDa, as well as NAD7 and NAD9, have been identified in the subcomplex by their cross-reactivity with heterologous antisera. The corresponding subunits in Neurospora are localized in a ‘peripheral arm’ of CI, which is known to assemble independently of a ‘membrane arm’. The maize NCS2 CI subcomplex is loosely bound to the membrane and is missing several subunits that could be membrane components. Thus, the mutant CI subcomplex may consist of a peripheral arm. A reduction in the steady-state levels of NAD7 and NAD9 in NCS2 mitochondria occurs despite normal rates of biosynthesis and there is a concomitant decrease of the nuclear encoded 76 kDa subunit. The reduction in CI-associated NADH dehydrogenase activity in the nad4 -deficient NCS2 mutant mitochondria is not associated with a compensatory increase in the activities or amounts of the putative ‘exogenous’ NAD(P)H dehydrogenases that are found in plant mitochondria.     

Mitochondrial complex I: A central regulator of the aging process

Mitochondria are considered major regulators of longevity, although their exact role in aging is not fully understood. Data from different laboratories show a negative correlation between reactive oxygen species (ROS) generated by complex I and lifespan. This suggests that complex I has a central role in the regulation of longevity. Here, we review data that both support and refute the role of complex I as a pacemaker of aging. We include data from our laboratory, where we have manipulated ROS production by the electron transport chain (ETC) in Drosophila melanogaster. The bypass of complex I increases the lifespan of the fruit fly, but it is not clear if this is caused by a reduction in ROS or by a change in the NAD+ to NADH ratio. We propose that complex I regulates aging through at least two mechanisms: (1) an ROS-dependent mechanism that leads to mitochondrial DNA damage and (2) an ROS-independent mechanism through the control of the NAD+ to NADH ratio. Control of the relative levels of NAD+ and NADH would allow the regulation of (1) glyco- and (2) lipoxidative-damage and (3) the activation of sirtuins.     

Extending Cassava Root Shelf Life via Reduction of Reactive Oxygen Species Production

One of the major constraints facing the large-scale production of cassava (Manihot esculenta) roots is the rapid postharvest physiological deterioration (PPD) that occurs within 72 h following harvest. One of the earliest recognized biochemical events during the initiation of PPD is a rapid burst of reactive oxygen species (ROS) accumulation. We have investigated the source of this oxidative burst to identify possible strategies to limit its extent and to extend cassava root shelf life. We provide evidence for a causal link between cyanogenesis and the onset of the oxidative burst that triggers PPD. By measuring ROS accumulation in transgenic low-cyanogen plants with and without cyanide complementation, we show that PPD is cyanide dependent, presumably resulting from a cyanide-dependent inhibition of respiration. To reduce cyanide-dependent ROS production in cassava root mitochondria, we generated transgenic plants expressing a codon-optimized Arabidopsis (Arabidopsis thaliana) mitochondrial alternative oxidase gene (AOX1A). Unlike cytochrome c oxidase, AOX is cyanide insensitive. Transgenic plants overexpressing AOX exhibited over a 10-fold reduction in ROS accumulation compared with wild-type plants. The reduction in ROS accumulation was associated with a delayed onset of PPD by 14 to 21 d after harvest of greenhouse-grown plants. The delay in PPD in transgenic plants was also observed under field conditions, but with a root biomass yield loss in the highest AOX-expressing lines. These data reveal a mechanism for PPD in cassava based on cyanide-induced oxidative stress as well as PPD control strategies involving inhibition of ROS production or its sequestration.     

死亡信号传递: 叶绿体与线粒体间信号交流调控植物程序性细胞死亡

程序性细胞死亡(PCD)是生物体受遗传调控的自主细胞死亡现象, 在植物生长发育和抵抗环境胁迫中起重要作用。PCD的发生可受线粒体中活性氧(ROS)诱导。中国科学院遗传与发育生物学研究所李家洋研究组早期的研究发现了1个拟南芥(Arabidopsis thaliana)细胞死亡突变体mod1, 并暗示植物细胞中存在叶绿体与线粒体之间的信号交流调控PCD, 但其中的具体作用机制尚不清楚。最近, 他们通过大规模筛选mod1突变体的抑制突变体, 克隆了3个新的抑制基因plNAD- MDH、DiT1和mMDH1。此3个基因分别编码质体定位的NAD依赖的苹果酸脱氢酶、叶绿体被膜定位的二羧酸转运蛋白1和线粒体定位的苹果酸脱氢酶1, 突变后都可抑制mod1中ROS的积累及PCD的发生。通过对这些基因进行深入的功能分析, 他们论证了苹果酸从叶绿体到线粒体的转运对线粒体中ROS的产生及随后PCD的诱导起重要作用。该研究拓展了我们对植物细胞中细胞器间交流的认识, 为我们深入理解植物PCD发生机制提供了新线索, 是该领域的一项突破性进展。     

Effects of hypoxia on relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in coronary arterial smooth muscle

Since controversy exists on how hypoxia influences vascular reactive oxygen species (ROS) generation, and our previous work provided evidence that it relaxes endothelium-denuded bovine coronary arteries (BCA) in a ROS-independent manner by promoting cytosolic NADPH oxidation, we examined how hypoxia alters relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in BCA. Methods were developed to image and interpret the effects of hypoxia on NAD(P)H redox based on its autofluorescence in the cytosolic, mitochondrial, and nuclear regions of smooth muscle cells isolated from BCA. Aspects of anaerobic glycolysis and cytosolic NADH redox in BCA were assessed from measurements of lactate and pyruvate. Imaging changes in mitosox and dehydroethidium fluorescence were used to detect changes in mitochondrial and cytosolic-nuclear superoxide, respectively. Hypoxia appeared to increase mitochondrial and decrease cytosolic-nuclear superoxide under conditions associated with increased cytosolic NADH (lactate/pyruvate), mitochondrial NAD(P)H, and hyperpolarization of mitochondria detected by tetramethylrhodamine methyl-ester perchlorate fluorescence. Rotenone appeared to increase mitochondrial NAD(P)H and superoxide, suggesting hypoxia could increase superoxide generation by complex I. However, hypoxia decreased mitochondrial superoxide in the presence of contraction to 30 mM KCl, associated with decreased mitochondrial NAD(P)H. Thus, while hypoxia augments NAD(P)H redox associated with increased mitochondrial superoxide, contraction with KCl reverses these effects of hypoxia on mitochondrial superoxide, suggesting mitochondrial ROS increases do not mediate hypoxic relaxation in BCA. Since hypoxia lowers pyruvate, and pyruvate inhibits hypoxia-elicited relaxation and NADPH oxidation in BCA, mitochondrial control of pyruvate metabolism associated with cytosolic NADPH redox regulation could contribute to sensing hypoxia.     

Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and cofactors across the inner mitochondrial membrane. In Saccharomyces cerevisiae, NAD+ is synthesized outside the mitochondria and must be imported across the permeability barrier of the inner mitochondrial membrane. However, no protein responsible for this transport activity has ever been isolated or identified. In this report, the identification and functional characterization of the mitochondrial NAD+ carrier protein (Ndt1p) is described. The NDT1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported NAD+ and, to a lesser extent, (d)AMP and (d)GMP but virtually not alpha-NAD+, NADH, NADP+, or NADPH. Transport was saturable with an apparent Km of 0.38 mM for NAD+. The Ndt1p-GFP was found to be targeted to mitochondria. Consistently with Ndt1p localization and its function as a NAD+ transporter, cells lacking NDT1 had reduced levels of NAD+ and NADH in their mitochondria and reduced activity of mitochondrial NAD+-requiring enzymes. Similar results were also found in the mitochondria of cells lacking NDT2 that encodes a protein (Ndt2p) displaying 70% homology with Ndt1p. The delta ndt1 delta ndt2 double mutant exhibited lower mitochondrial NAD+ and NADH levels than the single deletants and a more pronounced delay in growth on nonfermentable carbon sources. The main role of Ndt1p and Ndt2p is to import NAD+ into mitochondria by unidirectional transport or by exchange with intramitochondrially generated (d)AMP and (d)GMP.     

Nitric oxide accumulation is required to protect against iron-mediated oxidative stress in frataxin-deficient Arabidopsis plants

Frataxin is a mitochondrial protein that is conserved throughout evolution. In yeast and mammals, frataxin is essential for cellular iron (Fe) homeostasis and survival during oxidative stress. In plants, frataxin deficiency causes increased reactive oxygen species (ROS) production and high sensitivity to oxidative stress. In this work we show that a knock-down T-DNA frataxin-deficient mutant of Arabidopsis thaliana (atfh-1) contains increased total and organellar Fe levels. Frataxin deficiency leads also to nitric oxide (NO) accumulation in both, atfh-1 roots and frataxin null mutant yeast. Abnormally high NO production might be part of the defence mechanism against Fe-mediated oxidative stress.     

Regulation of NAD(H) pool and NADH/NAD(+) ratio by overexpression of nicotinic acid phosphoribosyltransferase for succinic acid production in Escherichia coli NZN111

Succinic acid is not the dominant fermentation product from glucose in wild-type Escherichia coli W1485. To reduce byproduct formation and increase succinic acid accumulation, pyruvate formate-lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, E. coli NZN111, the ldhA and pflB deletion strain, could not utilize glucose anaerobically due to the block of NAD(+) regeneration. To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase, a rate limiting enzyme of NAD(H) synthesis encoded by the pncB gene, resulted in a significant increase in cell mass and succinic acid production. Furthermore, the results indicated a significant increase in NAD(H) pool size, and decrease in the NADH/NAD(+) ratio from 0.64 to 0.13, in particular, the concentration of NAD(+) increased 6.2-fold during anaerobic fermentation. In other words, the supply of enough NAD(+) for NADH oxidation by regulation of NAD(H) salvage synthesis mechanism could improve the cell growth and glucose utilization anaerobically. In addition, the low NADH/NAD(+) ratio also change the metabolite distribution during the dual-phase fermentation. As a result, there was a significant increase in succinic acid production, and it is provided further evidence that regulation of NAD(H) pool and NADH/NAD(+) ratio was very important for succinic acid production.     

The decrease of NAD(P)H has a prominent role in dopamine toxicity

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.     

NAD(H)-dependent glutamate dehydrogenase is essential for the survival of Arabidopsis thaliana during dark-induced carbon starvation

Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.     

Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which is derived from NAD, have important roles as a redox carriers in metabolism. A combination of de novo and salvage pathways contribute to the biosynthesis of NAD in all organisms. The pathways and enzymes of the NAD salvage pathway in yeast and animals, which diverge at nicotinamide, have been extensively studied. Yeast cells convert nicotinamide to nicotinic acid, while mammals lack the enzyme nicotinamidase and instead convert nicotinamide to nicotinamide mononucleotide. Here we show that Arabidopsis thaliana gene At2g22570 encodes a nicotinamidase, which is expressed in all tissues, with the highest levels observed in roots and stems. The 244-residue protein, designated AtNIC1, converts nicotinamide to nicotinic acid and has a Km value of 118 +/- 17 microM and a Kcat value of 0.93 +/- 0.13 sec(-1). Plants homozygous for a null AtNIC1 allele, nic1-1, have lower levels of NAD and NADP under normal growth conditions, indicating that AtNIC1 participates in a yeast-type NAD salvage pathway. Mutant plants also exhibit hypersensitivity to treatments of abscisic acid and NaCl, which is correlated with their inability to increase the cellular levels of NAD(H) under these growth conditions, as occurs in wild-type plants. We also show that the growth of the roots of wild-type but not nic1-1 mutant plants is inhibited and distorted by nicotinamide.     

Arabidopsis DAL1 and DAL2, two RING finger proteins homologous to Drosophila DIAP1, are involved in regulation of programmed cell death

Programmed cell death (PCD) is a precise, genetically controlled cellular process with important roles in plant growth, development, and response to biotic and abiotic stress. However, the genetic mechanisms that control PCD in plants are unclear. Two Arabidopsis genes, DAL1 and DAL2 (for Drosophila DIAP1 like 1 and 2), encoding RING finger proteins with homology to DIAP1 were identified, and a series of experiments were performed to elucidate their roles in the regulation of PCD and disease resistance. Expression of DAL1 and DAL2 genes was induced in Arabidopsis plants after inoculation with virulent and avirulent strains of Pseudomonas syrinage pv. tomato (Pst) DC3000 or after infiltration with fumonisin B1 (FB1). Plants with mutations in the DAL1 and DAL2 genes displayed more severe disease after inoculation with an avirulent strain of Pst DC3000, but they showed similar disease severity as the wild-type plant after inoculation with a virulent strain of Pst DC3000. Significant accumulations of reactive oxygen species (ROS) and increased cell death were observed in the dal1 and dal2 mutant plants after inoculation with the avirulent strain of Pst DC3000. The dal mutant plants underwent extensive PCD upon infiltration of FB1 and displayed higher levels of ROS accumulation, callose deposition, and autofluorescence than the wild-type plants. Our data suggest that DAL1 and DAL2 may act as negative regulators of PCD in Arabidopsis.     

Plant succinic semialdehyde dehydrogenase: dissection of nucleotide binding by surface plasmon resonance and fluorescence spectroscopy

Recent kinetic studies revealed distinct modes of inhibition of mitochondrial Arabidopsis thaliana succinic semialdehyde dehydrogenase (At-SSADH1) by AMP and ATP. Inhibition of SSADH by ATP may represent an important mechanism of feedback regulation of the GABA shunt by the respiratory chain. Here we used two approaches to investigate the interaction of ATP with At-SSADH1. Cofactor displacement studies based on the reduced fluorescence intensity of free NADH versus that of enzyme-bound NADH revealed that both AMP and ATP decreased NADH-At-SSADH1 complex formation. The competitive inhibitor AMP displaced all bound NADH, while ATP, a noncompetitive inhibitor, could not, even in great excess, release all NADH from its binding site. To assess the effect of ATP on NAD-At-SSADH, we employed surface plasmon resonance to monitor nucleotide binding to immobilized At-SSADH1. For this, we used a Strep-tag II modified derivative of At-SSADH1 (designated ST-At-SSADH1). The tagged enzyme was tightly and reversibly captured by StrepTactin, which was covalently immobilized on a CM5 chip. The binding constants for NAD(+) and ATP were determined from titration curves and were in good agreement with the constants obtained from enzyme kinetics. Surface plasmon resonance measurements confirmed that ATP binds to a site different from the binding site for NAD(+). GTP competed with ATP. However, only ATP increased the dissociation constant of NAD(+) from SSADH. This explains the reduced affinity of NAD(+)/NADH to At-SSADH1 in the presence of ATP, as revealed by enzymatic kinetics, and supports our model of feedback regulation of SSADH and the GABA shunt by ATP.