SummaryRice tillering is an important agronomic trait for grain production. The HIGH-TILLERING DWARF1 (HTD1) gene encodes an ortholog of Arabidopsis MAX3. Complementation analyses for HTD1 confirm that the defect in HTD1 is responsible for both high-tillering and dwarf phenotypes in the htd1 mutant. The rescue of the Arabidopsis max3 mutant phenotype by the introduction of Pro 35S :HTD1 indicates HTD1 is a carotenoid cleavage dioxygenase that has the same function as MAX3 in synthesis of a carotenoid-derived signal molecule. The HTD1 gene is expressed in both shoot and root tissues. By evaluating Pro HTD1 :GUS expression, we found that the HTD1 gene is mainly expressed in vascular bundle tissues throughout the plant. Auxin induction of HTD1 expression suggests that auxin may regulate rice tillering partly through upregulation of HTD1 gene transcription. Restoration of dwarf phenotype after the removal of axillary buds indicates that the dwarfism of the htd1 mutant may be a consequence of excessive tiller production. In addition, the expression of HTD1, D3 and OsCCD8a in the htd1 and d3 mutants suggests a feedback mechanism may exist for the synthesis and perception of the carotenoid-derived signal in rice. Characterization of MAX genes in Arabidopsis, and identification of their orthologs in pea, petunia and rice indicates the existence of a conserved mechanism for shoot-branching regulation in both monocots and dicots.

No abstract

Usher syndrome type 2 (USH2) is the predominant form of USH, a leading genetic cause of combined deafness and blindness. PDZD7, a paralog of two USH causative genes, USH1C and USH2D (WHRN), was recently reported to be implicated in USH2 and non-syndromic deafness. It encodes a protein with multiple PDZ domains. To understand the biological function of PDZD7 and the pathogenic mechanism caused by PDZD7 mutations, we generated and thoroughly characterized a Pdzd7 knockout mouse model. The Pdzd7 knockout mice exhibit congenital profound deafness, as assessed by auditory brainstem response, distortion product otoacoustic emission and cochlear microphonics tests, and normal vestibular function, as assessed by their behaviors. Lack of PDZD7 leads to the disorganization of stereocilia bundles and a reduction in mechanotransduction currents and sensitivity in cochlear outer hair cells. At the molecular level, PDZD7 determines the localization of the USH2 protein complex, composed of USH2A, GPR98 and WHRN, to ankle links in developing cochlear hair cells, likely through its direct interactions with these three proteins. The localization of PDZD7 to the ankle links of cochlear hair bundles also relies on USH2 proteins. In photoreceptors of Pdzd7 knockout mice, the three USH2 proteins largely remain unchanged at the periciliary membrane complex. The electroretinogram responses of both rod and cone photoreceptors are normal in knockout mice at 1 month of age. Therefore, although the organization of the USH2 complex appears different in photoreceptors, it is clear that PDZD7 plays an essential role in organizing the USH2 complex at ankle links in developing cochlear hair cells. GenBank accession numbers: KF041446, KF041447, KF041448, KF041449, KF041450, KF041451.

A rice htd-1 mutant, related to tillering and dwarfing, was characterized. We show that the htd-1 mutant increases its tiller number by releasing axillary buds from dormant stage rather than by initiating more axillary buds. The dwarf is caused by averagely reducing each internode and panicle. Based on this dwarfing pattern, the htd-1 mutant could be grouped into dn-type dwarf defined by Takeda (Gamma Field Symp 16:1, 1977). In addition, the dwarfing of the htd-1 mutant was found independent of GA based on the analyses of two GA-mediated processes. Based on the quantitative determination of IAA and ABA and application of the two hormones exogenously to the seedlings, we inferred that the high tillering capacity of the htd-1 mutant should not be attributed to a defect in the synthesis of IAA or ABA. The genetic analysis of the htd-1 mutant indicated that the phenotypes of high tillering and dwarf were controlled by a recessive gene, termed htd1. By map-based cloning, the htd1 gene was fine mapped in a 30-kb DNA region on chromosome 4. Sequencing the target DNA region and comparing the counterpart DNA sequences between the htd-1 mutant and other rice varieties revealed a nucleotide substitution corresponding to an amino acid substitution from prolin to leucine in a predicted rice gene, OsCCD7, the rice orthologous gene of AtMAX3/CCD7. With the evidence of the association between the presence of one amino acid change in OsCCD7 and the abnormal phenotypes of the htd-1 mutant, OsCCD7 was identified as the candidate of the HTD1 gene.

Background: Assembly of the protein complex associated with Usher syndrome type 2 (USH2) is unclear. Results: WHRN and PDZD7 heterodimerization and their interactions with USH2A and GPR98 are required for USH2 complex formation. Conclusion: An USH2 quaternary protein complex may exist in vivo. Significance: This study provides clues to USH2 pathogenesis and may permit the complex reconstitution for therapeutic intervention.

Whirlin transgene recruits USH2A and VLGR1 to the PMC and is sufficient for the formation of the USH2 protein complex in photoreceptors. The combined hRK and AAV gene delivery system could be an effective gene therapy approach to treat retinal degeneration in USH2D patients.

Usher syndrome (USH) is the most common inherited deaf-blindness with the majority of USH causative genes also involved in nonsyndromic recessive deafness (DFNB). The mechanism underlying this disease variation of USH genes is unclear. Here, we addressed this issue by investigating the DFNB31 gene, whose mutations cause USH2D or DFNB31 depending on their position. We found that the mouse DFNB31 ortholog (Dfnb31) expressed different mRNA variants and whirlin protein isoforms in the cochlea and retina, where these isoforms played different roles spatially and temporally. Full-length (FL-) whirlin in photoreceptors and hair cell stereociliary bases is important for the USH type 2 protein complex, while FL- and C-terminal (C-) whirlins in hair cell stereociliary tips participate in stereociliary elongation. Mutations in the whirlin N-terminal region disrupted FL-whirlin isoform in the inner ear and retina but not C-whirlin in the inner ear, and led to retinal degeneration as well as moderate to severe hearing loss. By contrast, a mutation in the whirlin C-terminal region eliminated all normal whirlin isoforms but generated a truncated N-terminal whirlin protein fragment, which was partially functional in the retina and thus prevented retinal degeneration. Mice with this mutation had profound hearing loss. In summary, disruption of distinct whirlin isoforms by Dfnb31 mutations leads to a variety of phenotype configurations and may explain the mechanism underlying the different disease manifestations of human DFNB31 mutations. Our findings have a potential to improve diagnosis and treatment of USH disease and quality of life in USH patients.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.