The appropriate timing of flowering is pivotal for reproductive success in plants; thus, it is not surprising that flowering is regulated by complex genetic networks that are fine-tuned by endogenous signals and environmental cues. The Arabidopsis thaliana flowering-time gene SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) encodes a MADS box transcription factor and is one of the key floral activators integrating multiple floral inductive pathways, namely, long-day, vernalization, autonomous, and gibberellin-dependent pathways. To elucidate the downstream targets of SOC1, microarray analyses were performed. The analysis revealed that the soc1-2 knockout mutant has increased, and an SOC1 overexpression line has decreased, expression of cold response genes such as CBFs (for CRT/DRE binding factors) and COR (for cold regulated) genes, suggesting that SOC1 negatively regulates the expression of the cold response genes. By contrast, overexpression of cold-inducible CBFs caused late flowering through increased expression of FLOWERING LOCUS C (FLC), an upstream negative regulator of SOC1. Our results demonstrate the presence of a feedback loop between cold response and flowering-time regulation; this loop delays flowering through the increase of FLC when a cold spell is transient as in fall or early spring but suppresses the cold response when floral induction occurs through the repression of cold-inducible genes by SOC1.

The perception mechanism for the strigolactone (SL) class of plant hormones has been a subject of debate because their receptor, DWARF14 (D14), is an α/β-hydrolase that can cleave SLs. Here we show via time-course analyses of SL binding and hydrolysis by Arabidopsis thaliana D14, that the level of uncleaved SL strongly correlates with the induction of the active signaling state. In addition, we show that an AtD14D218A catalytic mutant that lacks enzymatic activity is still able to complement the atd14 mutant phenotype in an SL-dependent manner. We conclude that the intact SL molecules trigger the D14 active signaling state, and we also describe that D14 deactivates bioactive SLs by the hydrolytic degradation after signal transmission. Together, these results reveal that D14 is a dual-functional receptor, responsible for both the perception and deactivation of bioactive SLs.

The stage of gastrointestinal cancers has been correlated with the loss of heterozygosity (LOH) and the presence of microsatellite instability (MSI). This study delineated the category of the extent of LOH and the presence of MSI for the genetic classification of the intestinal-type and diffuse-type gastric cancers that frequently exhibited intralesional heterogeneity. A total of 390 tumor foci from 116 gastric cancers were screened using a panel of 40 microsatellite markers on chromosomes 3p, 4p, 5q, 8p, 9p, 13q, 17p, and 18q. One MSI-positive gastric cancer accompanying a LOH-positive focus and 19 gastric cancers with an intralesional LOH heterogeneity with a similar extent were identified. One hundred and sixteen gastric cancers were categorized based on the presence of MSI (16 cases) and the extent of LOH (100 cases) in a representative focus. A large fraction of MSI-positive cases was found in the intestinal-type (94%), late-onset (mean age 68 years), early-stage (75%) diseases (P<0.05). The diffuse-type gastric cancers with a baseline-level loss involving zero or one chromosome showed a correlation with the earlier onset (mean age 45 years), advanced-stage (81%) diseases (P<0.0001). In both the intestinal-type and diffuse-type gastric cancers, a low-level loss involving 0-3 chromosomes (2-3 chromosomes in the diffuse type) and a high-level loss involving 4-7 chromosomes were predominant in the early (69%) and advanced (86%) stages, respectively (P<0.0001), at similar mean ages of onset (61 years and 65 years). Gastric cancers were categorized into low-risk (MSI and low-level LOH) and high-risk (baseline-level and high-level LOH) genotypes displaying cell-type- and age-dependent oncogenicity.

Mutator phenotype tumors provide unique opportunities to unravel malignant progression because of various gene alterations acquired during clonal tumor evolution. Gastric carcinomas, which have been known to show frequent genetic instability, would be composed of initial gene alterations shared by most tumor areas and subsequent alterations restricted to particular tumor sites. To analyse the timing of genetic events, we examined separate sites of tumor tissue obtained from a given gastric carcinoma patient with microsatellite instability (MSI). Our study included 95 normal/tumor area pairs from 25 patients. Six of the 25 patients (24%) demonstrated various levels of MSI ranging from 7% (two of 30) to 97% (28 of 29) of markers tested in multiple tumor sites. Of the six patients, ®ve manifested frameshift mutations in a tract of ten deoxyadenosines within transforming growth factor b receptor type II and four demonstrated frameshift mutations in a tract of eight deoxyguanosines within BAX. These mutations were common to all tumor sites regardless of the various level of MSI phenotype, indicating initial events. Two of the six patients exhibited frameshift mutations in mononucleotide repeats of mismatch repair genes, hMSH3 and hMSH6, and the insulin-like growth factor II receptor in restricted tumor areas, indicating additional alterations. Insulin-like growth factor II receptor mutations appear to be caused by hMSH3 and hMSH6 mutations because the former mutations were con®ned to tumor portions with the latter two mismatch repair lesions. These results provide genetic progression evidence for gastric carcinomas of the mutator pathway. In this pathway, mismatch repair insuciency initially targets mononucleotide tracts of transforming growth factor b receptor type II and BAX. During tumorigenesis, primary mismatch repair failure may give rise to the secondary mismatch repair lesions, frameshift mutations of hMSH3 and hMSH6, which result in another tumorigenic mutation in the insulin-like growth factor II receptor.

Vernalization, a long-term cold-mediated acquisition of flowering competence, is critically regulated by VERNALIZATION INSENSITIVE 3 (VIN3), a gene induced by vernalization in Arabidopsis. Although the function of VIN3 has been extensively studied, how VIN3 expression itself is upregulated by long-term cold is not well understood. In the present study, we identified a vernalization-responsive cis-element in the VIN3 promoter, VREVIN3, composed of a G-box and an evening element (EE). Mutations in either the G-box or the EE prevented VIN3 expression from being fully induced upon vernalization, leading to defects in the vernalization response. We determined that the core clock proteins CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) associate with the EE of VREVIN3, both in vitro and in vivo. In a cca1 lhy double mutant background harboring a function FRIGIDA allele, long-term cold-mediated VIN3 induction and acceleration of flowering were impaired, especially under mild cold conditions such as at 12 °C. During prolonged cold exposure, oscillations of CCA1/LHY transcripts were altered, while CCA1 abundance increased at dusk, coinciding with the diurnal peak of VIN3 transcripts. We propose that modulation of the clock proteins CCA1 and LHY, participates in the systems involved in sensing long-term cold for the activation of VIN3 transcription.

Background: A loss of heterozygosity (LOH) represents a unilateral chromosomal loss that reduces the dose of highly repetitive Alu, L1, and LTR retroelements. The aim of this study was to determine if the LOH events can affect the spread of retroelement methylation in the 5'-end transitional area between the CpG islands and their nearest retroelements.

A key floral activator, FT, integrates stimuli from long-day, vernalization, and autonomous pathways and triggers flowering by directly regulating floral meristem identity genes in Arabidopsis (Arabidopsis thaliana). Since a small amount of FT transcript is sufficient for flowering, the FT level is strictly regulated by diverse genes. In this study, we show that WEREWOLF (WER), a MYB transcription factor regulating root hair pattern, is another regulator of FT. The mutant wer flowers late in long days but normal in short days and shows a weak sensitivity to vernalization, which indicates that WER controls flowering time through the photoperiod pathway. The expression and double mutant analyses showed that WER modulates FT transcript level independent of CONSTANS and FLOWERING LOCUS C. The histological analysis of WER shows that it is expressed in the epidermis of leaves, where FT is not expressed. Consistently, WER regulates not the transcription but the stability of FT mRNA. Our results reveal a novel regulatory mechanism of FT that is non cell autonomous.

Kinase Gcn2 is activated by amino acid starvation and downregulates translation initiation by phosphorylating the ␣ subunit of translation initiation factor 2 (eIF2␣). The Gcn2 kinase domain (KD) is inert and must be activated by tRNA binding to the adjacent regulatory domain. Previous work indicated that Saccharomyces cerevisiae Gcn2 latency results from inflexibility of the hinge connecting the N and C lobes and a partially obstructed ATP-binding site in the KD. Here, we provide strong evidence that a network of hydrophobic interactions centered on Leu-856 also promotes latency by constraining helix ␣C rotation in the KD in a manner relieved during amino acid starvation by tRNA binding and autophosphorylation of Thr-882 in the activation loop. Thus, we show that mutationally disrupting the hydrophobic network in various ways constitutively activates eIF2␣ phosphorylation in vivo and bypasses the requirement for a key tRNA binding motif (m2) and Thr-882 in Gcn2. In particular, replacing Leu-856 with any nonhydrophobic residue activates Gcn2, while substitutions with various hydrophobic residues maintain kinase latency. We further provide strong evidence that parallel, back-to-back dimerization of the KD is a step on the Gcn2 activation pathway promoted by tRNA binding and autophosphorylation. Remarkably, mutations that disrupt the L856 hydrophobic network or enhance hinge flexibility eliminate the need for the conserved salt bridge at the parallel dimer interface, implying that KD dimerization facilitates the reorientation of ␣C and remodeling of the active site for enhanced ATP binding and catalysis. We propose that hinge remodeling, parallel dimerization, and reorientation of ␣C are mutually reinforcing conformational transitions stimulated by tRNA binding and secured by the ensuing autophosphorylation of T882 for stable kinase activation.