Enhancement of Arabidopsis growth by non-24 hour day-night cycles.

Plant yield can be increased by matching the internal circadian rhythms with the external light and dark cycle (circadian resonance). The circadian resonance reported in the past was analyzed under light-dark cycles with 20-, 24-, or 28-hr periods; however, the mechanism for circadian resonance is still debatable due to the experimental time schedules in previous studies being few in number and widely separated. By analyzing the yield of Arabidopsis thaliana grown under eight different external light and dark periods, we found that the yield increased when the external cycle was 22 and 26 hr instead of 24 hr. Time course RNA-seq analysis determined that seedling circadian clock genes had a free-running period of 22 ± 1 hr. Furthermore, a group of genes with 25- to 26-hr period rhythms were also observed in the seedlings with a 22- ± 1-hr period as their circadian clock. We propose that resonance that occurred by matching the expression time of a group of genes with the 25- to 26-hr cycle and providing an external day-night cycle of 25 to 26 hr was one factor that caused the yield increase.

Prostaglandin E2 synchronizes lunar-regulated beach spawning in grass puffers.

Many organisms living along the coastlines synchronize their reproduction with the lunar cycle. At the time of spring tide, thousands of grass puffers (Takifugu alboplumbeus) aggregate and vigorously tremble their bodies at the water's edge to spawn. To understand the mechanisms underlying this spectacular semilunar beach spawning, we collected the hypothalamus and pituitary from male grass puffers every week for 2 months. RNA sequencing (RNA-seq) analysis identified 125 semilunar genes, including genes crucial for reproduction (e.g., gonadotropin-releasing hormone 1 [gnrh1], luteinizing hormone ß subunit [lhb]) and receptors for pheromone prostaglandin E (PGE). PGE2 is secreted into the seawater during the spawning, and its administration activates olfactory sensory neurons and triggers trembling behavior of surrounding individuals. These results suggest that PGE2 synchronizes lunar-regulated beach-spawning behavior in grass puffers. To further explore the mechanism that regulates the lunar-synchronized transcription of semilunar genes, we searched for semilunar transcription factors. Spatial transcriptomics and multiplex fluorescent in situ hybridization showed co-localization of the semilunar transcription factor CCAAT/enhancer-binding protein δ (cebpd) and gnrh1, and cebpd induced the promoter activity of gnrh1. Taken together, our study demonstrates semilunar genes that mediate lunar-synchronized beach-spawning behavior. VIDEO ABSTRACT.

Generation of Bradyrhizobium japonicum mutants with increased N2O reductase activity by selection after introduction of a mutated dnaQ gene.

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N(2)O) reductase (N(2)OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N(2)O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N(2)O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N(2)OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N(2)OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.

Gene expression of ascorbic acid-related enzymes in tobacco.

GDP-D-mannose pyrophosphorylase (GMPase) and L-galactono-1, 4-lactone dehydrogenase (GalLDH) are key enzymes in L-ascorbic acid (AsA) biosynthesis of plants, and a full-length cDNA for GMPase was isolated from tobacco using PCR. Additionally, expression of GMPase, GalLDH and other AsA-related enzymes was examined in tobacco tissues and cultured BY-2 cells, and the relationship between their expression patterns and AsA content is discussed. It was found that the expression of GalLDH and GMPase mRNAs was markedly suppressed by loading AsA, suggesting that AsA concentration in the cells may regulate AsA biosynthesis. Moreover, the expression of GMPase and GalLDH mRNAs in tobacco leaf also suggested that AsA biosynthesis may be induced by light.

Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for l-galactono-1,4-lactone dehydrogenase.

L-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative L-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco (Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content.