Moshe Sagi

Production of Reactive Oxygen Species by Plant NADPH Oxidases

NADPH oxidases (NOX) catalyze the production of superoxides, a type of reactive oxygen species (ROS). The dramatic induction of ROS production by human NOX2 in activated blood phagocytic cells and its role in promoting pathogen killing has long motivated research in this area ([Babior et al., 2002][

Plant respiratory burst oxidase homologs impinge on wound responsiveness and development in Lycopersicon esculentum.

Plant respiratory burst oxidase homologs (Rboh) are homologs of the human neutrophil pathogen-related gp91phox. Antisense technology was employed to ascertain the biological function of Lycopersicon esculentum (tomato) Rboh. Lines with diminished Rboh activity showed a reduced level of reactive oxygen species (ROS) in the leaf, implying a role for Rboh in establishing the cellular redox milieu. Surprisingly, the antisense plants acquired a highly branched phenotype, switched from indeterminate to determinate growth habit, and had fasciated reproductive organs. Wound-induced systemic expression of proteinase inhibitor II was compromised in the antisense lines, indicating that ROS intermediates supplied by Rboh are required for this wound response. Extending these observations by transcriptome analysis revealed ectopic leaf expression of homeotic MADS box genes that are normally expressed only in reproductive organs. In addition, both Rboh-dependent and -independent wound-induced gene induction was detected as well as transcript changes related to redox maintenance. The results provide novel insights into how the steady state cellular level of ROS is controlled and portrays the role of Rboh as a signal transducer of stress and developmental responses.

Diverse Subcellular Locations of Cryptogein-Induced Reactive Oxygen Species Production in Tobacco Bright Yellow-2 Cells

Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report real-time H2O2 accumulation in cell populations, we show that the internal signal for H2O2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H2O2 was also followed in individual cells using the 2′,7′-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H2O2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.

A critical role for ureides in dark and senescence-induced purine remobilization is unmasked in the Atxdh1 Arabidopsis mutant.

The remobilization of metabolites during stress and senescence plays an important role in optimal plant adaptation to the environment. The plant molybdenum co-factor (MoCo) and flavin-containing enzyme xanthine dehydrogenase (XDH; EC 1.2.1.37) are pivotal for purine remobilization, and catalyze the conversion of the purine catabolic products hypoxanthine and xanthine to uric acid, which is subsequently degraded to the ureides allantoin and allantoate. We observed that in wild-type plants conditions of extended darkness or increasing leaf age caused induction of transcripts related to purine catabolism, resulting in marked accumulation of the purine catabolic products allantoin and allantoate. In contrast, Arabidopsis mutants of XDH, Atxdh1, accumulated xanthine and showed premature senescence symptoms, as exemplified by enhanced chlorophyll degradation, extensive cell death and upregulation of senescence-related transcripts. When dark-treated mutant lines were re-exposed to light, they showed elevated levels of reactive oxygen species (ROS) and a higher mortality rate compared with wild-type plants. Interestingly, the level of ROS and mortality could be attenuated by the addition of allantoin and allantoate, suggesting that these metabolites can act as scavengers of ROS. The results highlight a crucial need for the controlled maintenance of ureide levels mediated by AtXDH1 activity during dark stress and ageing, and point to the dual functionality of ureides as efficient stores of nitrogen and as cellular protectants. Thus, the regulation of ureide levels by Atxdh1 has general implications for optimal plant survival during nutrient remobilization, such as occurs during normal growth, dark stress and senescence.

Hydrogen peroxide is a common signal for darkness- and ABA-induced stomatal closure in Pisum sativum

The requirement for hydrogen peroxide (H2O2) generation and action during stomatal closure induced by darkness and abscisic acid (ABA) was investigated in pea (Pisum sativum L.). Stomatal closure induced by darkness or ABA was inhibited by the H2O2-scavenging enzyme catalase or the antioxidant N-acetyl cysteine (NAC), or by diphenylene iodonium (DPI), an inhibitor of the H2O2-generating enzyme NADPH oxidase. Exogenous H2O2 induced stomatal closure in a dose- and time-dependent manner, and H2O2 was also required for ABA-inhibition of stomatal opening in the light. H2O2 accumulation in guard cells was increased by darkness or ABA, as assessed with the fluorescent dye dichlorodihydrofluorescein diacetate (H2-DCFDA) and confocal microscopy. Such increases were inhibited by catalase, NAC or DPI, consistent with the effects of these compounds on stomatal apertures. Employing polymerase chain reaction (PCR) with degenerate oligonucleotide primers, several NADPH oxidase homologues were identified from pea genomic DNA that had substantial identity to the Arabidopsis thaliana (L.) Heynh. rboh (respiratory burst oxidase homologue) genes. Furthermore, an antibody raised against the tomato rboh identified immunoreactive proteins in epidermal, mesophyll and guard cells.

Carbohydrate metabolism in leaves and assimilate partitioning in fruits of tomato (Lycopersicon esculentum L.) as affected by salinity

Abstract Salinity improve tomato fruit quality by enhancing hexose accumulation. To study the involvement of the carbohydrate influx into fruits under saline conditions, we have studied carbohydrate metabolism in leaves and the partitioning of photosynthetic 14 C in fruits of tomato plants exposed to 0, 50, or 100 mM NaCl. Carbohydrate content and the activity of ADP-Glc-PPase (EC 2.7.7.27) in the fruits were also determined at different stages of fruit development. Photosynthesis rates per unit leaf area were only slightly affected by moderate salinity (50 mM NaCl) while causing a considerable inhibition of leaf area expansion. Sucrose concentration was higher in leaves of plants exposed to NaCl than in non-saline controls, correlating with the enhanced activity of sucrose phosphate synthase (EC 2.3.1.14) and with the low activity of acid invertase (EC 3.2.1.26). Salinity enhanced the transport of 14 C-assimilates from the pulse leaf to adjacent fruits and the diversion of 14 C label to the starch fraction of the fruit. It also prolonged the period of starch accumulation in developing fruits. The immature fruit from salinity-treated plants showed significant higher activity of ADP-Glc-PPase with approximately twofold higher starch content than in controls. It is concluded that, under saline conditions, both a higher concentration of sucrose in the leaves and a faster rate of starch synthesis in the immature fruit may constitute part of a mechanism responsible for a higher sugar content in the mature fruit.

The Mo-hydroxylases xanthine dehydrogenase and aldehyde oxidase in ryegrass as affected by nitrogen and salinity

The influence of salinity and nitrogen source on xanthine dehydrogenase (XDH; EC 1.2.1.37) and aldehyde oxidase (AO; EC 1.2.3.1) was studied in annual ryegrass (Lolium multiflorum cv. Westerwoldicum). The activities of AO and XDH in the roots and shoots of ryegrass plants increased with salinity and NH 4 concentration. The salinity-enhanced activities of XDH and AO were more pronounced in the roots than in the shoots. Roots of NH 4 -grown plants had higher AO and XDH activities than plants grown in NO 3 . Immunoblotting revealed a higher level of AO protein in roots than in shoots. Root AO protein increased with salinity and was the highest in roots of NH 4 -grown plants. The assays of the molybdenum cofactor (MoCo) hydroxylases (XDH and AO) showed a similar response to salinity and nitrogen, and differed in molecular weight and substrate specificity. The concentration of ureides (allantoic acid and allantoin) increased with salinity and NH 4 , especially in the roots. The ureide contents of plants grown on NH 4 were higher than in plants receiving NO 3 . The increase in Mo-hydroxylases with salinity and NH 4 may constitute part of the mechanisms of plant adaptation to stress by (1) enhancing the activity of AO, which catalyzes the final step in biosynthesis of phytohormones such as abscisic acid (ABA), and (2) increased XDH activity and the subsequent production of ureides allowing transport of organic nitrogen compounds with a low C:N ratio.

Control of plant growth resides in the shoot, and not in the root, in reciprocal grafts of flacca and wild-type tomato (Lysopersicon esculentum), in the presence and absence of salinity stress

Grafted plants of flacca, an ABA-deficient mutant of tomato (Lycopersicon esculentum), and the wild-type variety Rheinlands Ruhm were grown with and without salinity stress to test the roles of roots and shoots in the regulation of plant growth. Fourteen days after exposure to 200 mM NaCl, shoot and root fresh weight, endogenous ABA concentrations, nitrate concentration, activities of selected enzymes related to nitrogen assimilation, and cation accumulation were determined. Rootstock genotype had little influence on the growth of the grafted plants, whereas grafted plants having wild-type shoots (Ws) produced more biomass than those having flacca shoots (Fs), irrespective of the salinity level. Growth of flacca shoots grafted onto wild-type rootstock (Fs/Wr) was superior to that of flacca shoots grafted onto flacca rootstock (Fs/Fr). The improved growth correlated with enhanced levels of ABA in the flaccashoots of Fs/Wr. In all the graft combinations, ABA content was higher in wild-type shoots than in flacca shoots, with or without salinity. There were no significant differences in root ABA concentrations among the different grafted types. Enhanced growth correlated with higher nitrate levels and higher nitrate reductase activity in the roots and shoots of plants with wild-type shoots and with higher shoot concentrations of ABA in plants with wild-type shoots. There were no significant differences in glutamine synthetase and phosphoenol pyruvate carboxylase activities in the shoots and roots of all the grafted plants, regardless of the salinity level. While shoot genotype determined the accumulation of K+ and Na+ in grafted plants regardless of salinity, it had no influence on Ca2+ concentrations. Regardless of the salinity, the total concentration of cations was the same in all the plants, while salinity decreased Mg2+ concentration in roots and shoots of all grafts, with the exception of flacca grafted shoots. The scion genotype – and its ABA level – thus played the major role in the growth of grafted plants, regardless of the rootstock genotype and the salinity of the growth medium.

Ammonium secretion by Colletotrichum coccodes activates host NADPH oxidase activity enhancing host cell death and fungal virulence in tomato fruits.

Colletotrichum pathogens of fruit and leaves are known ammonium secretors. Here, we show that Colletotrichum coccodes virulence, as measured by tomato (Solanum lycopersicum cv. Motelle) fruit tissue necrosis, correlates with the amount of ammonium secreted. Ammonium application to fruit tissue induced hydrogen peroxide (H(2)O(2)) accumulation. To examine whether the tomato NADPH oxidase, SlRBOH, is a source for the ammonium-induced H(2)O(2), wild-type and antisense lines abrogated for SlRBOH (SlRBOH-AS) were examined. Wild-type lines produced 7.5-fold more reactive oxygen species when exposed to exogenous ammonium than did SlRBOH-AS lines. C. coccodes colonization of wild-type tomato lines resulted in higher H(2)O(2) production and faster fungal growth rate compared with colonization in the SlRBOH-AS mutant, although the amount of ammonium secreted by the fungi was similar in both cases. Enhanced ion leakage and cell death of fruit tissue were correlated with H(2)O(2) accumulation, and treatment with the reactive oxygen scavenger N-acetyl-l-cysteine decreased H(2)O(2) production, ion leakage, and cell death. Importantly, the activation of reactive oxygen species production by ammonium was positively affected by an extracellular pH increase from 4 to 9, implying that ammonium exerts its control via membrane penetration. Our results show that C. coccodes activates host reactive oxygen species and H(2)O(2) production through ammonium secretion. The resultant enhancement in host tissue decay is an important step in the activation of the necrotrophic process needed for colonization.

Sulfite Reductase Protects Plants Against Sulfite Toxicity

Summary: Sulfite oxidase and the key elements of the sulfite network enzymes that include sulfite reductase, UDP-sulfoquinovose synthase, β-mercaptopyruvate sulfurtransferases, and adenosine-5′-phosphosulfate reductase has a important role in maintaining sulfite homeostasis, where sulfite appears to act as an orchestrating signal molecule. Plant sulfite reductase (SiR; Enzyme Commission 1.8.7.1) catalyzes the reduction of sulfite to sulfide in the reductive sulfate assimilation pathway. Comparison of SiR expression in tomato (Solanum lycopersicum ‘Rheinlands Ruhm’) and Arabidopsis (Arabidopsis thaliana) plants revealed that SiR is expressed in a different tissue-dependent manner that likely reflects dissimilarity in sulfur metabolism between the plant species. Using Arabidopsis and tomato SiR mutants with modified SiR expression, we show here that resistance to ectopically applied sulfur dioxide/sulfite is a function of SiR expression levels and that plants with reduced SiR expression exhibit higher sensitivity than the wild type, as manifested in pronounced leaf necrosis and chlorophyll bleaching. The sulfite-sensitive mutants accumulate applied sulfite and show a decline in glutathione levels. In contrast, mutants that overexpress SiR are more tolerant to sulfite toxicity, exhibiting little or no damage. Resistance to high sulfite application is manifested by fast sulfite disappearance and an increase in glutathione levels. The notion that SiR plays a role in the protection of plants against sulfite is supported by the rapid up-regulation of SiR transcript and activity within 30 min of sulfite injection into Arabidopsis and tomato leaves. Peroxisomal sulfite oxidase transcripts and activity levels are likewise promoted by sulfite application as compared with water injection controls. These results indicate that, in addition to participating in the sulfate assimilation reductive pathway, SiR also plays a role in protecting leaves against the toxicity of sulfite accumulation.