No abstract

The Cenozoic intramontane GongheGuide Basin in Qinghai Province, China, is tectonically controlled by the sinistral strikeslip framework of the Kunlun and Altyn Tagh-South Qilian faults in the northeastern Tibetan Plateau. The basin is fi lled with thick Cenozoic clastic sedimentary formations, which provide important evidence of the deformation of this part of the plateau, although they have long lacked good age constraints. Detailed magnetostratigraphic and paleontologic investigations of fi ve sections in the Guide Basin and their lithologic and sedimentary characteristics allow us to divide a formerly undifferentiated unit (the Guide Group) into six formations (where ages are now magnetostratigraphically well established, they are given in parentheses): the Amigang (1.8-2.6 Ma), Ganjia (2.6-3.6 Ma), and Herjia formations (3.6 to ca. 7.0-7.8 Ma), and the older Miocene Ashigong, Garang, and Guidemen formations. These rocks document a generally upward coarsening sequence, characterized by increasing accumulation rates. Increasing gravel content and sizes of its components, changes of bedding dips and source rock types, and marginal growth faults collectively refl ect accelerated deformation and uplift of the NE Tibetan Plateau after 8 Ma, punctuated by a sharp increase in sedimentation rate at ca. 3.2 Ma that refl ects the boulder conglomerates of the Ganjia formation. Interestingly, much of the vergence of the compressional deformation in the basin is to the south, accommodated by a sequence of six thrusts (F1-F6), which become active one by one progressively later toward the south, undoubtedly contributing to the uplift of this part of the plateau. F1 likely initiated the Guide Basin due to crustal fl exure in the Oligocene, F2 was active in the early Miocene, F4 and F5 at ca. 3.6 Ma, and F6 was active in the early Pleistocene. The detailed late Miocene and younger magnetostratigraphy allows us to place much improved time constraints on the deformation and, hence, uplift of northeastern Tibet, which, when compared with ages for events on other parts of the plateau, provides important boundary conditions for the geodynamical evolution of Tibet.

[1] The Xining subbasin of the Longzhong basin holds the longest continuous Cenozoic stratigraphic record at the margin of the northeastern Tibetan Plateau. Despite a rich biostratigraphic content (including the Xiejia mammal fauna), the tectonic evolution of the basin is largely unconstrained. In this study we present stratigraphic, biostratigraphic, and magnetostratigraphic results that provide a basis for reconstructing the Cenozoic tectonic evolution of the Xining basin with respect to adjacent regions of the northeastern Tibetan Plateau. Magnetostratigraphic analysis from three red bed sections in the Xining basin indicates continuous deposition at low and nearly constant accumulation rates (average 2.2 cm/kyr) from 52.0 to 17.0 Ma. We interpret this result to indicate that no major regional tectonic event implying large sediment accumulation variations has affected the Xining basin deposition during this considerable time window. In detail, accumulation rate variations outline a three-stage evolution with 1.8 cm/kyr from 52.0 Ma to 34.5 Ma, 4.1 cm/kyr from 34.5 to 31.0 Ma, and 2.3 cm/kyr from 31.0 to 17.0 Ma. The second-order increase between 34.5 and 31.0 Ma can be interpreted to result from either a distal tectonic event or to be of climatic origin. Although the region was tectonically quiescent for much of the Cenozoic, tectonic activity occurred during basin initiation (or reactivation) at circa 55.0-52.5 Ma and during intense basin deformation after 17.0 Ma.

Genome editing is one of the most powerful tools for revealing gene function and improving crop plants. Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been used as a powerful and efficient tool for genome editing in various organisms. Here, we report genome editing in tobacco (Nicotiana tabacum) mediated by the CRISPR/Cas9 system. Two genes, NtPDS and NtPDR6, were used for targeted mutagenesis. First, we examined the transient genome editing activity of this system in tobacco protoplasts, insertion and deletion (indel) mutations were observed with frequencies of 16.2-20.3% after transfecting guide RNA (gRNA) and the nuclease Cas9 in tobacco protoplasts. The two genes were also mutated using multiplexing gRNA at a time. Additionally, targeted deletions and inversions of a 1.8-kb fragment between two target sites in the NtPDS locus were demonstrated, while indel mutations were also detected at both the sites. Second, we obtained transgenic tobacco plants with NtPDS and NtPDR6 mutations induced by Cas9/gRNA. The mutation percentage was 81.8% for NtPDS gRNA4 and 87.5% for NtPDR6 gRNA2. Obvious phenotypes were observed, etiolated leaves for the psd mutant and more branches for the pdr6 mutant, indicating that highly efficient biallelic mutations occurred in both transgenic lines. No significant off-target mutations were obtained. Our results show that the CRISPR/Cas9 system is a useful tool for targeted mutagenesis of the tobacco genome.

Aquaporins are water channel proteins that facilitate the passage of water through biological membranes and play a crucial role in plant growth. We show that ethylene treatment significantly reduced petal size, inhibited expansion of petal abaxial subepidermal cells, and decreased petal water content in rose (Rosa hybrida 'Samantha'). Here, we report the isolation of a plasma membrane aquaporin (PIP) gene, Rh-PIP2;1, and characterized its potential role in ethylene-inhibited petal expansion. Rh-PIP2;1 is mainly localized on the plasma membrane and belongs to the class 2 subfamily of PIP proteins. We show that Rh-PIP2;1 is an active water channel. The transcripts of Rh-PIP2;1 are highly abundant in petal epidermal cells, especially in the abaxial subepidermal cells. The expression of Rh-PIP2;1 is highly correlated with petal expansion and tightly down-regulated by ethylene. Furthermore, we demonstrate that in Rh-PIP2;1-silenced flowers, petal expansion was greatly inhibited and anatomical features of the petals were similar to those of ethylene-treated flowers. We argue that Rh-PIP2;1 plays an important role in petal cell expansion and that ethylene inhibits petal expansion of roses at least partially by suppressing Rh-PIP2;1 expression.

Flowering time and an ability to tolerate abiotic stresses are important for plant growth and development. We characterized BBX24, a zinc finger transcription factor gene, from Chrysanthemum morifolium and found it to be associated with both flowering time and stress tolerance. Transgenic lines with suppressed expression of Cm-BBX24 (Cm-BBX24-RNAi) flowered earlier than wild-type plants and showed decreased tolerance to freezing and drought stresses. Global expression analysis revealed that genes associated with both photoperiod and gibberellin (GA) biosynthesis pathways were upregulated in Cm-BBX24-RNAi lines, relative to the wild type. By contrast, genes that were upregulated in overexpressing lines (Cm-BBX24-OX), but downregulated in Cm-BBX24-RNAi lines (both relative to the wild type), included genes related to compatible solutes and carbohydrate metabolism, both of which are associated with abiotic stress. Cm-BBX24 expression was also influenced by daylength and GA 4/7 application. Under long days, changes in endogenous GA 1 , GA 4 , GA 19 , and GA 20 levels occurred in young leaves of transgenic lines, relative to the wild type. Regulation of flowering involves the FLOWERING TIME gene, which integrates photoperiod and GA biosynthesis pathways. We postulate that Cm-BBX24 plays a dual role, modulating both flowering time and abiotic stress tolerance in chrysanthemum, at least in part by influencing GA biosynthesis.

Dehydration inhibits petal expansion resulting in abnormal flower opening and results in quality loss during the marketing of cut flowers. We constructed a suppression subtractive hybridization library from rose (Rosa hybrida) flowers containing 3,513 unique expressed sequence tags and analyzed their expression profiles during cycles of dehydration. We found that 54 genes were up-regulated by the first dehydration, restored or even down-regulated by rehydration, and once again up-regulated by the second dehydration. Among them, we identified a putative NAC family transcription factor (RhNAC2). With transactivation activity of its carboxyl-terminal domain in yeast (Saccharomyces cerevisiae) cell and Arabidopsis (Arabidopsis thaliana) protoplast, RhNAC2 belongs to the NAC transcription factor clade related to plant development in Arabidopsis. A putative expansin gene named RhEXPA4 was also dramatically up-regulated by dehydration. Silencing RhNAC2 or RhEXPA4 in rose petals by virusinduced gene silencing significantly decreased the recovery of intact petals and petal discs during rehydration. Overexpression of RhNAC2 or RhEXPA4 in Arabidopsis conferred strong drought tolerance in the transgenic plants. RhEXPA4 expression was repressed in RhNAC2-silenced rose petals, and the amino-terminal binding domain of RhNAC2 bound to the RhEXPA4 promoter. Twenty cell wall-related genes, including seven expansin family members, were up-regulated in Arabidopsis plants overexpressing RhNAC2. These data indicate that RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals and that RhEXPA4 expression may be regulated by RhNAC2.

Accumulation of indigestible lipofuscin and decreased mitochondrial energy production are characteristic age-related changes of post-mitotic retinal pigment epithelial (RPE) cells in the human eye. To test whether these two forms of age-related impairment have interdependent effects, we quantified the ATPdependent phagocytic function of RPE cells loaded or not with the lipofuscin component A2E and inhibiting or not mitochondrial ATP synthesis either pharmacologically or genetically. We found that physiological levels of lysosomal A2E reduced mitochondrial membrane potential and inhibited oxidative phosphorylation (OXPHOS) of RPE cells. Furthermore, in media with physiological concentrations of glucose or pyruvate, A2E significantly inhibited phagocytosis. Antioxidants reversed these effects of A2E, suggesting that A2E damage is mediated by oxidative processes. Because mitochondrial mutations accumulate with aging, we generated novel genetic cellular models of RPE carrying mitochondrial DNA point mutations causing either moderate or severe mitochondrial dysfunction. Exploring these mutant RPE cells we found that, by itself, only the severe but not the moderate OXPHOS defect reduces phagocytosis. However, sub-toxic levels of lysosomal A2E are sufficient to reduce phagocytic activity of RPE with moderate OXPHOS defect and cause cell death of RPE with severe OXPHOS defect. Taken together, RPE cells rely on OXPHOS for phagocytosis when the carbon energy source is limited. Our results demonstrate that A2E accumulation exacerbates the effects of moderate mitochondrial dysfunction. They suggest that synergy of sub-toxic lysosomal and mitochondrial changes in RPE cells with age may cause RPE dysfunction that is known to contribute to human retinal diseases like agerelated macular degeneration. Retinal pigment epithelial (RPE)5 cells form a polarized monolayer epithelium between the photoreceptors of the neurosensory retina and the choroidal capillary bed. Daily phagocytosis of outer segment (OS) tips shed by adjacent photoreceptors is a vital task of the RPE (recently reviewed by Strauss (1)). RPE cells are post-mitotic and face each ϳ30 photoreceptor outer segments in the human eye, all of which shed their distal tip containing stacked membrane disks once a day. Diurnal phagocytosis and digestion of thousands of OS disks for life renders RPE cells the most active phagocytes in the body. Photoreceptor function strictly depends on efficient RPE phagocytosis of spent OS. Complete failure of RPE cells to engulf OS causes rapid photoreceptor degeneration in the Royal College of Surgeons rat (2-4). Impaired RPE phagocytosis also contributes to human retinal disease such as retinitis pigmentosa and, likely, age-related macular degeneration (5, 6).The continuous nature of outer segment renewal implies that any delay in OS removal by aged or damaged RPE will gradually cause OS components to accumulate. RPE cells are at risk for oxidative damage due to their location in the highly oxygenated environment of the outer retina and th...