Cross-talk between the cellular redox state and the circadian system in Neurospora.

The circadian system is composed of a number of feedback loops, and multiple feedback loops in the form of oscillators help to maintain stable rhythms. The filamentous fungus Neurospora crassa exhibits a circadian rhythm during asexual spore formation (conidiation banding) and has a major feedback loop that includes the FREQUENCY (FRQ)/WHITE COLLAR (WC) -1 and -2 oscillator (FWO). A mutation in superoxide dismutase (sod)-1, an antioxidant gene, causes a robust and stable circadian rhythm compared with that of wild-type (Wt). However, the mechanisms underlying the functions of reactive oxygen species (ROS) remain unknown. Here, we show that cellular ROS concentrations change in a circadian manner (ROS oscillation), and the amplitudes of ROS oscillation increase with each cycle and then become steady (ROS homeostasis). The ROS oscillation and homeostasis are produced by the ROS-destroying catalases (CATs) and ROS-generating NADPH oxidase (NOX). cat-1 is also induced by illumination, and it reduces ROS levels. Although ROS oscillation persists in the absence of frq, wc-1 or wc-2, its homeostasis is altered. Furthermore, genetic and biochemical evidence reveals that ROS concentration regulates the transcriptional function of WCC and a higher ROS concentration enhances conidiation banding. These findings suggest that the circadian system engages in cross-talk with the cellular redox state via ROS-regulatory factors.

Global warming, plant paraquat resistance, and light signal transduction through nucleoside diphosphate kinase as a paradigm for increasing food supply.

Light signal transduction was studied in extracts of mycelia of the fungus Neurospora crassa, and the third internodes of dark-grown Pisum sativum cv Alaska. Both processes increased the phosphorylation of nucleoside diphosphate kinase (NDPK). NDPK may function as a carrier of reduction equivalents, as it binds NADH, thereby providing electrons to transform singlet oxygen to superoxide by catalases (CAT). As the C-termini of NDPK interact with CAT which receive singlet oxygen, emitted from photoreceptors post light perception (which is transmitted to ambient triplet oxygen), we hypothesize that this may increase phospho-NDPK. Singlet oxygen, emitted from the photoreceptor, also reacts with unsaturated fatty acids in membranes thereby forming malonedialdehyde, which in turn could release ions from, e.g., the thylacoid membrane thereby reducing the rate of photosynthesis. A mutant of Alaska pea, which exhibited two mutations in chloroplast NDPK-2 and one mutation in mitochondrial localized NDPK-3, was resistant to reactive oxygen species including singlet oxygen and showed an increase in the production of carotenoids, anthocyanine, and thereby could reduce the concentration of singlet oxygen. The reduction of the concentration of singlet oxygen is predicted to increase the yield of crop plants, such as Alaska pea, soybean, rice, wheat, barley, and sugarcane. This approach to increase the yield of crop plants may contribute not only to enhance food supply, but also to reduce the concentration of CO(2) in the atmosphere.

ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants.

This study investigated the reactive oxygen species (ROS) tolerance mechanism of a paraquat-resistant Pisum sativum line (R3-1) compared with the wild type (WT). Physiological and biochemical analyses showed significant differences in the phenotypes, such as delayed leaf and floral development, superior branching, and greater biomass and yields in the R3-1 line, as well as an increased level of antioxidant pigments and a lower rate of cellular lipid peroxidation in the resistant R3-1. Additionally, the phosphorylation of crude proteins showed distinguishable differences in band mobility and intensity between the R3-1 and WT plants. cDNA cloning and sequence analysis of NDPKs, which were candidate phosphorylated proteins, revealed that two of the deduced amino acids in NDPK2 (IL12L and Glu205Lys) and one in NDPK3 (P45S) were mutated in R3-1. Using glutathione S-transferase-NDPK fusion constructs, we found that the precursor recombinant R3-1 NDPK2 showed an increased level of activity and autophosphorylation in R3-1 plants compared to WT plants. Native PAGE analysis of the crude proteins revealed that NDPK and catalase (CAT) activity co-existed in the same area of the gel. In a yeast two-hybrid assay, the N-terminal region of NDPK2 showed an interaction with the full-length CAT1 protein. Furthermore, we found that WT showed a decreased level of CAT activity compared with R3-1 under illumination and/or on media containing ROS-releasing reagents. Taken together, these results suggest that there is a strong interaction between NDPK2 and CAT1 in R3-1 plants, which possibly plays a vital role in the antioxidant defense against ROS.

Nucleoside diphosphate kinase-1 regulates hyphal development via the transcriptional regulation of catalase in Neurospora crassa.

A nucleoside diphosphate kinase-1-disrupted (ndk-1(RIP-1)) mutant was observed to be defective in aerial hyphal and conidial development. In this study, two types of hyphae, fine and thick, were observed in wild-type (Wt) strains. However, only fine-type hyphae were observed in the ndk-1(RIP-1) mutants. The ndk-1(RIP-1) mutants were stimulated by oxidative stress and constitutively expressed an antioxidant enzyme catalase (CAT)-3. Furthermore the ndk-1(RIP-1) mutants could form thick hyphae by oxidative stress and a disruption of cat-3. These results suggest that the loss of thick hyphae in the ndk-1(RIP-1) mutants may be caused by the over-expression of cat-3.

Conidiation rhythm and light entrainment in superoxide dismutase mutant in Neurospora crassa.

Conidial formation in the filamentous fungus Neurospora crassa is regulated by nutritional conditions, light, and the circadian clock. We found that a sod-1 mutant, with a defective superoxide dismutase catalyzing the conversion of superoxide to hydrogen peroxide, had a slightly shorter period length than the wild type and clear conidial banding similar to a mutant of band (bd). However, unlike the bd mutant, the sod-1 mutant could sustain conidial banding with light pulses on a nutrient-rich medium, which involved an enhancement of the light-induced transcription of frequency (frq). sod-1 was hypersensitive to entrainment of the conidiation rhythm by light in race tubes. Furthermore, a frq(10); sod-1 double mutant showed conidiation rhythm in darkness and could be synchronized to light/dark cycles by the masking effect of light. These genetic analyses suggested that intracellular reactive oxygen species (ROS) act on circadian conidiation via multiple circadian clocks and output pathways.

Catalase-1 (CAT-1) and nucleoside diphosphate kinase-1 (NDK-1) play an important role in protecting conidial viability under light stress in Neurospora crassa.

Recently we reported that Catalase-1 (CAT-1) played an important role in protecting conidial viability in Neurospora crassa, and interacted with a light signal transducer, nucleoside diphosphate kinase-1 (NDK-1). To disclose the functional interaction between CAT-1 and NDK-1 at the genetic level, we created CAT-1 and NDK-1 double mutants, cat-1;ndk-1-1 and cat-1;ndk-1-2, by crossing single mutants of cat-1 ( RIP ) and ndk-1 ( P72H ) previously isolated in our laboratory. The double mutant strains grew normally, but showed increased CAT-2 activity. In cat-1 ( RIP ), NDK activity was increased when dCDP was used as a substrate. ndk-1 ( P72H ), cat-1;ndk-1-1, and cat-1;ndk-1-2 were more sensitive to riboflavin than the wild type and cat-1 ( RIP ) under strong light (100 microE m(-2) s(-1)). The pull-down experiment suggests that His-tagged NDK-1 is bound to [(32)P]NADH. However, his-tagged NDK-1(P72H) was not bound to [(32)P]NADH. The double mutants showed much lower conidial viability and lost all conidial germination ability much more rapidly than cat-1 ( RIP ), when they were cultured under continuous light for more than 2 weeks. These results indicate that the interaction of CAT-1 with NDK-1 plays an important role in supporting the survival of conidia under oxidative and light-induced stress including singlet oxygen, and confirm our former conclusion that reactive oxygen species play an important role in light signal transduction via NDK-1 at the genetic level.

Loss of Catalase-1 (Cat-1) results in decreased conidial viability enhanced by exposure to light in Neurospora crassa.

Light is one of the most important factors inducing morphogenesis in Neurospora crassa. The reception of light triggers the generation of reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)). Catalase-1 (Cat-1) is one of three catalases known to detoxify H(2)O(2) into water and oxygen. We reported that the photomorphogenetic characteristics of mutants in nucleoside diphosphate kinase-1 (NDK-1), a light signal transducer, are severely affected, and NDK-1 interacted with Cat-1 in a yeast two-hybrid assay. To disclose the function of Cat-1, we created a Cat-1 loss-of-function mutant (cat-1 ( RIP )) by the repeat induced point-mutation (RIPing) method. No Cat-1 activity was detected in the mutant strain. Forty guanines were replaced with adenines in the cat-1 gene of cat-1 ( RIP ), which caused 30 amino acid substitutions. The mutant strain grew normally, but its conidia and mycelia were more sensitive to H(2)O(2) than those of the wild type. The lack of Cat-1 activity also caused a significant reduction in the conidial germination rate. Furthermore, light enhanced this reduction in cat-1 ( RIP ) more than that in the wild type. Introduction of cat-1 into the mutant reversed all of these defective phenotypes. These results indicate that Cat-1 plays an important role in supporting the survival of conidia under oxidative and light-induced stress.

Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa.

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. In Neurospora crassa, NDK-1 is suggested to control catalases in response to heat, oxidative stress and light. In this study, we identified the presence of NDK-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fractions in the mycelial cell. A light-dependent localization of NDK-1 was shown by Western blotting and immunohistochemical analysis using anti-NDK-1 antibody. In the mycelia, NDK-1 was compartmentalized on the plasma membrane in darkness, while it was relocated in the cytoplasm under light. These results suggest that NDK-1 protein was translocated from the plasma membrane to cytoplasm in response to light, and may interact with catalase.

Interaction of nucleoside diphosphate kinase and catalases for stress and light responses in Neurospora crassa.

Nucleoside diphosphate kinase (NDK) is an ubiquitous enzyme with the function of a signal transducer. In Neurospora crassa, an ndk-1(P72H) mutant carrying the point mutation Pro72His was isolated. We found that ndk-1(P72H) showed hypersensitivity to oxidative and heat stress and a decrease in the levels of catalase (Cat)-1 and -3 induced by oxidative, heat stress and illumination compared with wild type (Wt). We found, by conducting a yeast two-hybrid assay, that Cat-1 interacted with NDK-1. NDK-1 was suggested to control Cat-1 and Cat-3 at the post-transcriptional level in response to heat, oxidative stress and light.

Photomorphogenetic characteristics are severely affected in nucleoside diphosphate kinase-1 (ndk-1)-disrupted mutants in Neurospora crassa.

We previously demonstrated that the NDK-1 (Nucleoside Diphosphate Kinase-1) point mutant, ndk-1(P72H), displays a defective phenotype in light-induced perithecial polarity in Neurospora crassa. To investigate the biological function of NDK-1 in detail, we isolated two ndk-1 mutants, ndk-1(RIP-1) and ndk-1(RIP-2), using the RIPing (repeat induced point mutation) method. Notably, we detected no accumulation of ndk-1(RIP-1) mRNA and truncated NDK-1(RIP-2) protein. The ndk-1(RIP) mutants exhibited altered morphogenesis; (1) aerial hypha was not formed with no conidium formation, (2) the mutants exhibited colonial, and very slow mycelial growth on a solid medium and by shaking culture in a liquid medium, (3) light-induced carotenoid accumulation in mutant mycelia is reduced to less than half that by wild type, (4) the mutants exhibited spiral growth of mycelia, and (5) female sterility with defective protoperithecium formation. The morphogenetic processes of 1, 3, and 5 are light induced in the wild type. Moreover, despite only 10-20% of total nucleoside diphosphate kinase activity, the accumulation of relevant transcripts in the ndk-1(RIP) mutants, such as al-1 and al-2, was similar to that of wild type.

Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa.

Previously, we found that intracellular reactive oxygen species (ROS) affect photomorphogenesis in Neurospora crassa. In this study, we investigated the physiological roles of ROS in the response to light and found that the exposure of mycelia to air was important for the light-induced carotenogenesis. Mycelia treated with a high concentration of O(2) gas and H(2)O(2) to release ROS showed an enhancement of light-induced carotenoid accumulation and the expression of gene related to light-inducible carotenogenesis. These results suggested that stimuli caused by the exposure of the mycelia to air containing O(2) gas triggered the light-induced carotenoid synthesis.

Molecular cloning and characterization of nucleoside diphosphate (NDP) kinases from Chinese cabbage (Brassica campestris).

Nucleoside diphosphate kinases (NDPKs) are key metabolic enzymes that catalyze the synthesis of non-adenosine nucleoside triphosphates (NTP) by transfer of the terminal phosphate between NDP and NTP. Recently we isolated three NDPK cDNAs from Chinese cabbage cDNA library. BcNDK1 has 636 bp and encodes a putative 17.4 kDa protein, BcNDK2 has 854 bp and encodes a putative 25.5 kDa protein, and BcNDK3 is 986 bp long and encodes a putative 25.7 kDa protein. The precursor proteins of BcNDK2 and BcNDK3 have long N-terminal extensions containing putative chloroplast or mitochondrial targeting sequences. A phylogenic tree showed that the 3 BcNDKs are highly homologous to other plant NDPK genes, especially those of Arabidopsis. Expression of the BcNDK genes depended on the developmental stage and the conditions of seed germination. Most notably, expression of BcNDK2 increased dramatically in seedlings transferred to the light after germinating in the dark. In addition, BcNDK3 differed from BcNDK1 in being highly expressed in the hooks and cotyledons of seedlings. Although all BcNDKs were highly expressed in petals, BcNDK1 was also expressed in pistils. Expression of each of the BcNDKs increased as the flower bud matured. These results indicate that NDPKs are involved in physiological pathways activated by a variety of environmental conditions and at different developmental stages.

Reactive oxygen species affect photomorphogenesis in Neurospora crassa.

In Neurospora crassa, several biological phenomena such as the synthesis of carotenoids in the mycelia and polarity of perithecia are regulated by light. We found that a sod-1 mutant, with a defective Cu,Zn-type superoxide dismutase (SOD), showed accelerated light-dependent induction of carotenoid accumulation in the mycelia compared with the wild type. The initial rate of light-induced carotenoid accumulation in the sod-1 mutant was faster than that in the vvd mutant known to accumulate high concentrations. This acceleration was suppressed by treatment with antioxidant reagents. Light-induced transcription of genes involved in carotenoid synthesis, al-1, -2, and -3, was sustained in the sod-1 mutant, whereas it was transient in the wild type. Moreover sod-1 was defective in terms of light-induced polarity of perithecia. By genetic analysis, the enhancement in light-inducible carotenoid synthesis in sod-1 was dependent on the wild type alleles of wc-1 and wc-2. However, the sod-1;vvd double mutant showed additive effects on the carotenoid accumulation in the mycelia. These results suggested that intracellular reactive oxygen species regulated by SOD-1 could affect the light-signal transduction pathway via WC proteins.

Arabidopsis NDK1 is a component of ROS signaling by interacting with three catalases.

Plants sense various environmental stimuli and have specific signaling pathways to respond to these cues. We focused on light responsive components and found that NDKs were phosphorylated specifically after red light irradiation in Pisum sativum [Tanaka et al. (1998) J. Photochem. Photobiol. B 45: 113] and after blue light irradiation in Neurospora crassa [Oda and Hasunuma (1997) Mol. Gen. Genet. 256: 593, Ogura et al. (2001) J. Biol. Chem. 276: 21228]. We performed yeast two-hybrid screening using AtNDK1, the counterpart of NDK-P1 (Pisum sativum NDK1) in Arabidopsis, as bait, and isolated catalase3 (AtCat3). Interactions between AtNDK1-AtCAT1 and AtNDK1-AtCAT2 were also detected with the two-hybrid system. Non-denaturing two-dimensional gel electrophoresis of crude extracts from plants revealed that catalase and NDK activities co-migrated in the same area of the gel. Transgenic plants expressing AtNDK1 under control of the CaMV 35S promoter exhibited tolerance to paraquat and high ability to eliminate exogenous H2O2. These results indicate that AtNDK1 has a role in ROS response.

Putative functions of nucleoside diphosphate kinase in plants and fungi.

The putative functions of NDP (nucleoside diaphosphate) kinases from various organisms focusing to fungi and plants are described. The biochemical reactions catalyzed by NDP kinase are as follows. (i) Phosphotransferring activity from mainly ATP to cognate NDPs generating nucleoside triphosphates (NTPs). (ii) Autophosphorylation activity from ATP and GTP. (iii) Protein kinase (phosphotransferring) activity phosphorylating such as myelin basic protein. NDP kinase could function to provide NTPs as a housekeeping enzyme. However, recent works proved possible functions of the NDP kinases in the processes of signal transduction in various organisms, as described below. 1) By use of the extracts of the mycelia of a filamentous fungus Neurospora crassa blue-light irradiation could increase the phosphorylation of a 15-kDa protein, which was purified and identified to be NDP kinase (NDK-1). By use of the etiolated seedlings of Pisum sativum cv Alaska and Oryza sativa red-light irradiation of intact plants increased the phosphorylation of NDP kinase. However, successive irradiation by red-far-red reversed the reaction, indicating that phytochrome-mediated light signals are transduced to the phosphorylation of NDP kinase. 2) NDP kinase localizing in mitochondria is encoded by nuclear genome and different from those localized in cytoplasm. NDP kinase in mitochondria formed a complex with succinyl CoA synthetase. 3) In Spinicia oleraceae two different NDP kinases were detected in the chloroplast, and in Pisum sativum two forms of NDP kinase originated from single species of mRNA could be detected in the choloroplast. However, the function of NDP kinases in the choloroplast is not yet known. 4) In Neurospora crassa a Pro72His mutation in NDP kinase (ndk-1Pro72His) deficient in the autophosphorylation and protein kinase activity resulted in lacking the light-induced polarity of perithecia. In wild-type directional light irradiation parallel to the solid medium resulted in the formation of the perithecial beak at the top of perithecia, which was designated as "light-induced polarity of perithecia." In wild-type in darkness the beak was formed at random places on perithecia, and in ndkPro72His mutant the perithecial beak was formed at random places even under directional light illumination. The introduction of genomic DNA and cDNA for ndk-1 demonstrated that the wild-type DNAs suppressed the mutant phenotype. With all these results except for the demonstration in Neurospora, most of the phenomena are elusive and should be solved in the molecular levels concerning with NDP kinases.