). † These authors contributed equally to this work. SUMMARYLOV KELCH PROTEIN2 (LKP2), ZEITLUPE (ZTL)/LOV KELCH PROTEIN1 (LKP1) and FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1) constitute a family of Arabidopsis F-box proteins that regulate the circadian clock. Over-expression of LKP2 or ZTL causes arrhythmicity of multiple clock outputs under constant light and in constant darkness. Here, we show the significance of LKP2 and ZTL in the photoperiodic control of flowering time in Arabidopsis. In plants over-expressing LKP2, CO and FT expression was down-regulated under longday conditions. LKP2 and ZTL physically interacted with FKF1, which was recruited from the nucleus into cytosolic speckles. LKP2 and ZTL inhibited the interaction of FKF1 with CYCLING DOF FACTOR 1, a ubiquitination substrate for FKF1 that is localized in the nucleus. The Kelch repeat regions of LKP2 and ZTL were sufficient for their physical interaction with FKF1 and translocation of FKF1 to the cytoplasm. Overexpression of LKP2 Kelch repeats induced late flowering under long-day conditions. lkp2 ztl double mutant plants flowered earlier than wild-type plants under short-day (non-inductive) conditions, and both CO and FT expression levels were up-regulated in the double mutant plants. The early flowering of lkp2 ztl was dependent on FKF1. LKP2, ZTL or both affected the accumulation of FKF1 protein during the early light period. These results indicate that an important role of LKP2 and ZTL in the photoperiodic pathway is repression of flowering under non-inductive conditions, and this is dependent on FKF1.

Monoubiquitination is a major histone post-translational modification. In humans, the histone H2B K120 and histone H4 K31 residues are monoubiquitinated and may form transcriptionally active chromatin. In this study, we reconstituted nucleosomes containing H2B monoubiquitinated at position 120 (H2Bub120) and/or H4 monoubiquitinated at position 31 (H4ub31). We found that the H2Bub120 and H4ub31 monoubiquitinations differently affect nucleosome stability: the H2Bub120 monoubiquitination enhances the H2A–H2B association with the nucleosome, while the H4ub31 monoubiquitination decreases the H3–H4 stability in the nucleosome, when compared with the unmodified nucleosome. The H2Bub120 and H4ub31 monoubiquitinations both antagonize the Mg2+-dependent compaction of a poly-nucleosome, suggesting that these monoubiquitinations maintain more relaxed conformations of chromatin. In the crystal structure, the H2Bub120 and H4ub31 monoubiquitinations do not change the structure of the nucleosome core particle and the ubiquitin molecules were flexibly disordered in the H2Bub120/H4ub31 nucleosome structure. These results revealed the differences and similarities of the H2Bub120 and H4ub31 monoubiquitinations at the mono- and poly-nucleosome levels and provide novel information to clarify the roles of monoubiquitination in chromatin.

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Sweetpotato roots were stored under a continuous flow of 0% or 1% O 2 (balance N 2 ) or air for 7 days at 20°C to study the effects of short-term exposure to low O 2 on their physiological responses and quality. During the course of the experiment, no visible signs of injury or decay were observed. However, low O 2 treatments increased the soluble solid content and weak off-odors were detected by olfactory evaluation in roots stored at 0% O 2 . The intensity of off-odors increased as the concentrations of acetaldehyde and ethanol increased in roots during storage. Ethanol concentrations were higher than those of acetaldehyde, which remained low during storage in 1% O 2 and air, but increased greatly in roots stored at 0% O 2 . Pyruvate decarboxylase (PDC) activities in roots exposed to 0% or 1% O 2 increased by 3.1-and 2-fold respectively over levels in roots stored in air by day 7. Alcohol dehydrogenase (ADH) activities in roots exposed to 0% or 1% O 2 increased by 1.6-and 1.7-fold respectively over levels in roots stored in air by day 7. ADH-specific activity was about 10-times that of PDC. The pH of root homogenate exposed to air remained constant, whereas the pH increased and decreased, respectively, in roots stored at 0% or 1% O 2 . PDC showed stability over the pH range 5.5-7.0, whereas ADH exhibited stability over the pH range 6.0-7.5. The Km of PDC in sweetpotato was 0.56 mM for pyruvate, whereas the Km of ADH was 0.19 mM for acetaldehyde. From these results, there may be some potential for the short-term exposure of sweetpotato roots to low O 2 in place of low temperature treatment to prolong shelf-life, although ethanol fermentation may be accelerated under low O 2 atmospheres.

Purpose: We investigated the potential of baseline 4′-[methyl-11C]-thiothymidine ([ 11C]4DST) PET for predicting loco-regional control with head and neck squamous cell carcinoma (HNSCC). Methods: A retrospective analysis was performed using volumetric parameters, such as SUVmax, proliferative tumor volume (PTV), and total legion proliferation (TLP), of pretreatment [11C]4DST PET for 91 patients with HNSCC with primary lesions in the oral cavity, hypopharynx, supraglottis, and oropharynx. As for the oropharynx, p16-negative cases were included. PTV and TLP were calculated for primary lesions and metastatic lymph nodes combined. We examined the association among the parameters and recurrence-free survival (RFS) and whether case selection focused on biological characteristics improved the accuracy of prognosis prediction. Results: The area under the curve (AUCs) using PTV and TLP for the oropharyngeal/hypopharyngeal/supraglottis groups were high (0.91 and 0.87, respectively), whereas that of SUVmax was 0.66 (p < 0.01). On the other hand, the oral group had lower AUCs for PTV and TLP at 0.72 and 0.77. When all cases were examined, the AUC values using PTV and TLP were 0.84 and 0.83, respectively. Conclusion: Baseline [11C]4DST PET/CT volume-based parameters can provide important prognostic information with p16-negative oropharyngeal, hypopharyngeal, and supraglottic cancer patients.

Alternative splicing is an important mechanism in the process of eukaryotic nuclear mRNA precursors producing multiple protein products from a single gene. Although group I self-splicing introns usually perform regular splicing, limited examples of alternative splicing have also been reported. The exon-skipping type of splicing has been observed in genes containing two group I introns. To characterize splicing patterns (exon-skipping/exon-inclusion) of tandemly aligned group I introns, we constructed a reporter gene containing two Tetrahymena introns flanking a short exon. To control splicing patterns, we engineered the two introns in a pairwise manner to design pairs of introns that selectively perform either exon-skipping or exon-inclusion splicing. Through pairwise engineering and biochemical characterization, the structural elements important for the induction of exon-skipping splicing were elucidated.

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