A double-negative feedback loop formed by the morning genes CIRCADIAN CLOCK ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY) and the evening gene TIMING OF CAB EXPRESSION1 (TOC1) contributes to regulation of the circadian clock in Arabidopsis. A 24-h circadian cycle starts with the peak expression of CCA1 at dawn. Although CCA1 is targeted by multiple transcriptional repressors, including PSEUDO-RESPONSE REGULATOR9 (PRR9), PRR7, PRR5 and CCA1 HIKING EXPEDITION (CHE), activators of CCA1 remain elusive. Here we use mathematical modelling to infer a co-activator role for LIGHT-REGULATED WD1 (LWD1) in CCA1 expression. We show that the TEOSINTE BRANCHED 1-CYCLOIDEA-PCF20 (TCP20) and TCP22 proteins act as LWD-interacting transcriptional activators. The concomitant binding of LWD1 and TCP20/TCP22 to the TCP-binding site in the CCA1 promoter activates CCA1. Our study reveals activators of the morning gene CCA1 and provides an action mechanism that ensures elevated expression of CCA1 at dawn to sustain a robust clock.

Background: MK-7 is a highly valuable vitamin K2 produced by Bacillus subtilis. Biofilm forming caused by quorum sensing (QS) system was beneficial for MK-7 synthesis. However, the specific regulator in QS system which play the most significant role in biofilm formation and MK production have not been revealed at genetic level, and this limits the possibility of further increasing MK-7 production.Results: In this study, the influence of different mutants in QS system on biofilm formation and MK-7 production was investigated. The transcriptional regulator SinR was chosen finally because the complete knocking out of sinR (KO-SinR) maximized the biofilm biomass and increased the yield of MK-7. However, KO-SinR led to a large number of spore and wrinkle forming, which slow down even stop the MK-7 biosynthesis. Five SinR mutants (E97K, Y101L, W104K, R105S, SinRquad) were constructed by site-directed mutagenesis of four aromatic residues (Glu97, Tyr101, Trp104 and Arg105). Among these, SinRquad formed more wrinkly but smooth biofilm, and obtained the maximum MK-7 value (102.56±2.84 mg/L), which was ten times of the parent strain. Comparative transcriptional analysis showed that SinRquad promoted the synthesis of extracellular polymeric substances and inhibited the swimming motility of late-flagellar. In addition, SinRquad upregulated the expression level of ctaC-G operator and qcrA-C operator which encode the cytochromes. Upregulation of components in the oxidative respiratory chain of B. subtilis was due to the upregulation of NADH dehydrogenases. Most NADH dehydrogenases especially sdhA-C and glpD, increased 1.01-, 3.93-, 1.87-, 1.11- fold, respectively. The increase in expression level of NADH dehydrogenases indicated the ratio of NADH/NAD+ decreased and more electrons produced for the electron transport system (ETM). Wrinkly and smooth biofilm formed a network of interconnected channels with low resistance to liquid flow was beneficial to obtain a more stable state of electron transport chain, which facilitate the metabolic flux of four modules of MK-7 synthesis pathways.Conclusions: In this study, we report for the first time that SinRquad has significant effects on the MK-7 synthesis by forming wrinkly and smooth biofilm, upregulating the expression level of most NADH dehydrogenases, providing more stable state of the electron transport.