A Small-Molecule Modulator Affecting the Clock-Associated PSEUDO-RESPONSE REGULATOR 7 Amount.

Circadian clocks are biological timekeeping systems that coordinate genetic, metabolic and physiological behaviors with the external day-night cycle. The clock in plants relies on the transcriptional-translational feedback loops transcription-translation feedback loop (TTFL), consisting of transcription factors including PSUEDO-RESPONSE REGULATOR (PRR) proteins, plant lineage-specific transcriptional repressors. Here, we report that a novel synthetic small-molecule modulator, 5-(3,4-dichlorophenyl)-1-phenyl-1,7-dihydro-4H-pyrazolo[3,4-d] pyrimidine-4,6(5H)-dione (TU-892), affects the PRR7 protein amount. A clock reporter line of Arabidopsis was screened against the 10,000 small molecules in the Maybridge Hitfinder 10K chemical library. This screening identified TU-892 as a period-lengthening molecule. Gene expression analyses showed that TU-892 treatment upregulates CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) mRNA expression. TU-892 treatment reduced the amount of PRR7 protein, a transcriptional repressor of CCA1. Other PRR proteins including TIMING OF CAB EXPRESSION 1 were altered less by TU-892 treatment. TU-892-dependent CCA1 upregulation was attenuated in mutants impaired in PRR7. Collectively, TU-892 is a novel type of clock modulator that reduces the levels of PRR7 protein.

Improvement of recombinant miraculin production in transgenic tomato by crossbreeding-based genetic background modification.

An important optimization step in plant-based recombinant protein production systems is the selection of an appropriate cultivar after a potential host has been determined. Previously, we have shown that transgenic tomatoes of the variety 'Micro-Tom' accumulate incredibly high levels of miraculin (MIR) due to the introduction of MIR gene controlled by a CaMV35S promoter and a heat-shock protein terminator. However, 'Micro-Tom' is unsuitable for commercial production of MIR as it is a dwarf cultivar characterized by small-sized fruit and poor yield. Here, we used the crossbreeding approach to transfer the high MIR accumulation trait of transgenic 'Micro-Tom' tomatoes to 'Natsunokoma' and 'Aichi First', two commercial cultivars producing medium and large fruit sizes, respectively. Fruits of the resultant crossbred lines were larger (~ 95 times), but their miraculin accumulation levels (~ 1,062 µg/g fresh mass) were comparable to the donor cultivar, indicating that the high miraculin accumulation trait was preserved regardless of fruit size or cultivar. Further, the transferred trait resulted in a 3-4 fold increase in overall miraculin production than that of the previously reported line 5B. These findings demonstrate the effectiveness of crossbreeding in improving MIR production in tomatoes and could pave the way for a more efficient production of recombinant proteins in other plants.

Structure-Function Study of a Novel Inhibitor of Cyclin-Dependent Kinase C in Arabidopsis.

The circadian clock, an internal time-keeping system with a period of about 24 h, coordinates many physiological processes with the day-night cycle. We previously demonstrated that BML-259 [N-(5-isopropyl-2-thiazolyl) phenylacetamide], a small molecule with mammal CYCLIN DEPENDENT KINASE 5 (CDK5)/CDK2 inhibition activity, lengthens Arabidopsis thaliana (Arabidopsis) circadian clock periods. BML-259 inhibits Arabidopsis CDKC kinase, which phosphorylates RNA polymerase II in the general transcriptional machinery. To accelerate our understanding of the inhibitory mechanism of BML-259 on CDKC, we performed structure-function studies of BML-259 using circadian period-lengthening activity as an estimation of CDKC inhibitor activity in vivo. The presence of a thiazole ring is essential for period-lengthening activity, whereas acetamide, isopropyl and phenyl groups can be modified without effect. BML-259 analog TT-539, a known mammal CDK5 inhibitor, did not lengthen the period nor did it inhibit Pol II phosphorylation. TT-361, an analog having a thiophenyl ring instead of a phenyl ring, possesses stronger period-lengthening activity and CDKC;2 inhibitory activity than BML-259. In silico ensemble docking calculations using Arabidopsis CDKC;2 obtained by a homology modeling indicated that the different binding conformations between these molecules and CDKC;2 explain the divergent activities of TT539 and TT361.

Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period.

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional-translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

The accumulation of recombinant miraculin is independent of fruit size in tomato.

The taste-modifying protein miraculin (MIR) has received increasing interest as a new low-calorie sweetener. In our previous study using the tomato variety 'Micro-Tom,' it was shown that in transgenic tomatoes in which MIR was expressed by using the cauliflower mosaic virus 35S promoter (p35S) and a heat shock protein terminator (tHSP) cassette (p35S-MIR-tHSP), higher levels of miraculin accumulated than when MIR was driven by the nopaline synthase terminator (tNOS) cassette (p35S-MIR-tNOS). 'Micro-Tom' is a dwarf tomato used for research and shows a low yield. To achieve high productivity of MIR, it is essential to improve the MIR accumulation potential by using high-yielding cultivars. In this study, we evaluate whether the high MIR accumulation trait mediated by the tHSP appears even when fruit size increases. A line in which the p35S-MIR-tHSP cassette was introduced into a high-yielding variety was bred by backcrossing. The line homozygous for MIR showed higher accumulation of MIR than the heterozygous line. Despite large differences in fruit size, the MIR level in the backcross line was similar to that in the p35S-MIR-tHSP line (background 'Micro-Tom'). It was approximately 3.1 times and 4.0 times higher than those in miracle fruits and the p35S-MIR-tNOS tomato line 5B ('Moneymaker' background, which exhibits the highest miraculin productivity achieved thus far), respectively. These results demonstrate that the high MIR accumulation trait mediated by the tHSP appears even when fruit size is increased.

3,4-Dibromo-7-Azaindole Modulates Arabidopsis Circadian Clock by Inhibiting Casein Kinase 1 Activity.

The circadian clock is a timekeeping system for regulation of numerous biological daily rhythms. One characteristic of the circadian clock is that period length remains relatively constant in spite of environmental fluctuations, such as temperature change. Here, using the curated collection of in-house small molecule chemical library (ITbM chemical library), we show that small molecule 3,4-dibromo-7-azaindole (B-AZ) lengthened the circadian period of Arabidopsis thaliana (Arabidopsis). B-AZ has not previously been reported to have any biological and biochemical activities. Target identification can elucidate the mode of action of small molecules, but we were unable to make a molecular probe of B-AZ for target identification. Instead, we performed other analysis, gene expression profiling that potentially reveals mode of action of molecules. Short-term treatment of B-AZ decreased the expression of four dawn- and morning-phased clock-associated genes, CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1), LATE ELONGATED HYPOCOTYL (LHY), PSEUDO-RESPONSE REGULATOR 9 (PRR9) and PRR7. Consistently, amounts of PRR5 and TIMING OF CAB EXPRESSION 1 (TOC1) proteins, transcriptional repressors of CCA1, LHY, PRR9 and PRR7 were increased upon B-AZ treatment. B-AZ inhibited Casein Kinase 1 family (CK1) that phosphorylates PRR5 and TOC1 for targeted degradation. A docking study and molecular dynamics simulation suggested that B-AZ interacts with the ATP-binding pocket of human CK1 delta, whose amino acid sequences are highly similar to those of Arabidopsis CK1. B-AZ-induced period-lengthening effect was attenuated in prr5 toc1 mutants. Collectively, this study provides a novel and simple structure CK1 inhibitor that modulates circadian clock via accumulation of PRR5 and TOC1.

Structure-function study of a novel inhibitor of the casein kinase 1 family in Arabidopsis thaliana.

Casein kinase 1 (CK1) is an evolutionarily conserved protein kinase family among eukaryotes. Studies in non-plants have shown CK1-dependent divergent biological processes, but the collective knowledge regarding the biological roles of plant CK1 lags far behind other members of the Eukarya. One reason for this is that plants have many more genes encoding CK1 than do animals. To accelerate our understanding of the plant CK1 family, a strong CK1 inhibitor that efficiently inhibits multiple members of the CK1 protein family in vivo (i.e., in planta) is required. Here, we report a novel, specific, and effective CK1 inhibitor in Arabidopsis. Using circadian period-lengthening activity as an estimation of the CK1 inhibitor effect in vivo, we performed a structure-activity relationship study of analogues of the CK1 inhibitor PHA767491 (1,5,6,7-tetrahydro-2-(4-pyridinyl)-4H-pyrrolo[3,2-c]pyridin-4-one hydrochloride). A propargyl group at the pyrrole nitrogen atom (AMI-212) or a bromine atom at the pyrrole C3 position (AMI-23) had stronger CK1 inhibitory activity than PHA767491. A hybrid molecule of AMI-212 and AMI-23 (AMI-331) was about 100-fold more inhibitory than the parent molecule PHA767491. Affinity proteomics using an AMI-331 probe showed that the targets of AMI-331 inhibition are mostly CK1 kinases. As such, AMI-331 is a potent and selective CK1 inhibitor that shows promise in the research of CK1 in plants.