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.

Dual role of BKI1 and 14-3-3 s in brassinosteroid signaling to link receptor with transcription factors.

The plasma membrane-localized plant steroid hormone receptor, BRASSINOSTEROID INSENSITIVE 1 (BRI1), is quiescent in the absence of steroids, largely due to a negative regulator, BRI1 KINASE INHIBITOR 1 (BKI1). Here, we report that the steroid-induced, plasma membrane-dissociated and phosphorylated BKI1 also plays positive roles in BR signaling by interacting with a subset of 14-3-3 proteins. The cytosolic fraction of BKI1 carboxyl terminal region enhances BR signaling. Mutations of two serine residues in this region lead to reduced phosphorylation by the BRI1 kinase and constitutive plasma membrane localization. The 14-3-3 proteins can interact with the phosphorylated BKI1 through a motif that contains the two phosphorylation sites to release inhibition of BRI1 by BKI1. Meanwhile, the cytosolic BKI1 antagonizes the 14-3-3 s and enhances accumulation of BRI1 EMS SUPPRESSOR 1 (BES1)/BRASSINAZOLE RESISTANT 1 (BZR1) in the nucleus to regulate BR-responses.

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.

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.

[Construction of expression vector with antisense Rubisco activase gene and its genetic transformation in rice].

In this research, Rubisco activase gene (rca) was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRI after confirming by sequencing. The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHI and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02. The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice (Oryza. Sativa L.ssp.japonica) cultivar Zhonghua11 via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin. Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T0 genomes and inherited to T1. The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO2 concentration, even could not grow. The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls, and their contents of RCA and Rubisco changed significantly. These plants generated in this experiment will be used to study the relationship between RCA and Rubisco and their regulation.