Identification of a Bipotential Precursor Cell in Hepatic Cell Lines Derived from Transgenic Mice Expressing Cyto-Met in the Liver

Met murine hepatocyte (MMH) lines were established from livers of transgenic mice expressing constitutively active human Met. These lines harbor two cell types: epithelial cells resembling the parental populations and flattened cells with multiple projections and a dispersed growth habit that are designated palmate. Epithelial cells express the liver-enriched transcription factors HNF4 and HNF1α, and proteins associated with epithelial cell differentiation. Treatments that modulate their differentiation state, including acidic FGF, induce hepatic functions. Palmate cells show none of these properties. However, they can differentiate along the hepatic cell lineage, giving rise to: (a) epithelial cells that express hepatic transcription factors and are competent to express hepatic functions; (b) bile duct-like structures in three-dimensional Matrigel cultures. Derivation of epithelial from palmate cells is confirmed by characterization of the progeny of individually fished cells. Furthermore, karyotype analysis confirms the direction of the phenotypic transition: palmate cells are diploid and the epithelial cells are hypotetraploid. The clonal isolation of the palmate cell, an immortalized nontransformed bipotential cell that does not yet express the liver-enriched transcription factors and is a precursor of the epithelial-hepatocyte in MMH lines, provides a new tool for the study of mechanisms controlling liver development

Chromatin-associated regulation of sorbitol synthesis in flower buds of peach

[EN] Key message PpeS6PDH gene is postulated to mediate sorbitol synthesis in flower buds of peach concomitantly with specific chromatin modifications. Abstract Perennial plants have evolved an adaptive mechanism involving protection of meristems within specialized structures named buds in order to survive low temperatures and water deprivation during winter. A seasonal period of dormancy further improves tolerance of buds to environmental stresses through specific mechanisms poorly known at the molecular level. We have shown that peach PpeS6PDH gene is down-regulated in flower buds after dormancy release, concomitantly with changes in the methylation level at specific lysine residues of histone H3 (H3K27 and H3K4) in the chromatin around the translation start site of the gene. PpeS6PDH encodes a NADPH-dependent sorbitol-6-phosphate dehydrogenase, the key enzyme for biosynthesis of sorbitol. Consistently, sorbitol accumulates in dormant buds showing higher PpeS6PDH expression. Moreover, PpeS6PDH gene expression is affected by cold and water deficit stress. Particularly, its expression is up-regulated by low temperature in buds and leaves, whereas desiccation treatment induces PpeS6PDH in buds and represses the gene in leaves. These data reveal the concurrent participation of chromatin modification mechanisms, transcriptional regulation of PpeS6PDH and sorbitol accumulation in flower buds of peach. In addition to its role as a major translocatable photosynthate in Rosaceae species, sorbitol is a widespread compatible solute and cryoprotectant, which suggests its participation in tolerance to environmental stresses in flower buds of peach.This work was funded by the Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA)-FEDER (RF2013-00043-C02-02) and the Ministry of Science and Innovation of Spain (AGL2010-20595). AL was funded by a fellowship co-financed by the European Social Fund and the Instituto Valenciano de Investigaciones Agrarias (IVIA).Lloret, A.; Martinez Fuentes, A.; Agustí Fonfría, M.; Badenes, ML.; Rios, G. (2017). Chromatin-associated regulation of sorbitol synthesis in flower buds of peach. Plant Molecular Biology. 95(4-5):507-517. https://doi.org/10.1007/s11103-017-0669-6S507517954-5Andersen CL, Jensen JL, Ørntoft TF (2004) Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 64:5245–5250. doi: 10.1158/0008-5472.CAN-04-0496Bai S, Saito T, Ito A et al (2016) Small RNA and PARE sequencing in flower bud reveal the involvement of sRNAs in endodormancy release of Japanese pear (Pyrus pyrifolia ‘Kosui’). BMC Genomics 17:230. doi: 10.1186/s12864-016-2514-8Bielenberg DG, Wang Y, Li Z et al (2008) Sequencing and annotation of the evergrowing locus in peach (Prunus persica [L.] Batsch) reveals a cluster of six MADS-box transcription factors as candidate genes for regulation of terminal bud formation. Tree Genet Genomes 4:495–507. doi: 10.1007/s11295-007-0126-9Bieleski RL (1969) Accumulation and translocation of sorbitol in apple phloem. Aust J Biol Sci 22:611–620. doi: 10.1071/BI9690611Bieleski RL (1982) Sugar alcohols. In: Loewus F, Tanner W (eds) Encyclopedia of plant physiology, new series 13A. Springer-Verlag, Berlin, pp 158–192Bortiri E, Oh SH, Gao FY, Potter D (2002) The phylogenetic utility of nucleotide sequences of sorbitol 6-phosphate dehydrogenase in Prunus (Rosaceae). Am J Bot 89:1697–1708. doi: 10.3732/ajb.89.10.1697Chouard P (1960) Vernalization and its relations to dormancy. Annu Rev Plant Physiol 11:191–238. doi: 10.1146/annurev.pp.11.060160.001203Conde D, Le Gac AL, Perales M et al (2017) Chilling-responsive DEMETER-LIKE DNA demethylase mediates in poplar bud break. Plant Cell Environ 40:2236–2249. doi: 10.1111/pce.13019Couvillon GA, Erez A (1985) Influence of prolonged exposure to chilling temperatures on bud break and heat requirement for bloom of several fruit species. J Amer Soc Hort Sci 110:47–50de la Fuente L, Conesa A, Lloret A, Badenes ML, Ríos G (2015) Genome-wide changes in histone H3 lysine 27 trimethylation associated with bud dormancy release in peach. Tree Genet Genomes 11:45. doi: 10.1007/s11295-015-0869-7Deng W, Buzas DM, Ying H et al (2013) Arabidopsis polycomb repressive complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes. BMC Genomics 14:593. doi: 10.1186/1471-2164-14-593Escobar-Gutiérrez AJ, Gaudillère JP (1996) Distribution, metabolism and role of sorbitol in higher plants—A review. Agronomie 16:281–298. doi: 10.1051/agro:19960502Escobar-Gutiérrez AJ, Zipperlin B, Carbonne F, Moing A, Gaudillére JP (1998) Photosynthesis, carbon partitioning and metabolite content during drought stress in peach seedlings. Aust J Plant Physiol 25:197–205. doi: 10.1071/PP97121Eshghi S, Tafazoli E, Dokhani S, Rahemi M, Emam Y (2007) Changes in carbohydrate contents in shoot tips, leaves and roots of strawberry (Fragaria x ananassa Duch) during flower-bud differentiation. Sci Hortic 113:255–260. doi: 10.1016/j.scienta.2007.03.014Everard JD, Cantini C, Grumet R, Plummer J, Loescher WH (1997) Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery. Plant Physiol 113:1427–1435. doi: 10.1104/pp.113.4.1427Fennell A (2014) Genomics and functional genomics of winter low temperature tolerance in temperate fruit crops. Crit Rev Plant Sci 33:125–140. doi: 10.1080/07352689.2014.870410Figueroa CM, Iglesias AA (2010) Aldose-6-phosphate reductase from apple leaves: importance of the quaternary structure for enzyme activity. Biochimie 92:81–88. doi: 10.1016/j.biochi.2009.09.013Gao M, Tao R, Miura K, Dandekar AM, Sugiura A (2001) Transformation of Japanese persimmon (Diospyros kaki Thunb) with apple cDNA encoding NADP-dependent sorbitol-6-phosphate dehydrogenase. Plant Sci 160:837–845. doi: 10.1016/S0168-9452(00)00458-1Grant CR, ap Rees T (1981) Sorbitol metabolism by apple seedlings. Phytochemistry 20:1505–1511. doi: 10.1016/S0031-9422(00)98521-2Hartman MD, Figueroa CM, Arias DG, Iglesias AA (2017) Inhibition of recombinant aldose-6-phosphate reductase from peach leaves by hexose-phosphates, inorganic phosphate and oxidants. Plant Cell Physiol 58:145–155. doi: 10.1093/pcp/pcw180Horvath DP, Anderson JV, Chao WS, Foley ME (2003) Knowing when to grow: signals regulating bud dormancy. Trends Plant Sci 8:534–540. doi: 10.1016/j.tplants.2003.09.013Horvath DP, Sung S, Kim D, Chao W, Anderson J (2010) Characterization, expression and function of DORMANCY ASSOCIATED MADS-BOX genes from leafy spurge. Plant Mol Biol 73:169–179. doi: 10.1007/s11103-009-9596-5Hussain S, Niu Q, Yang F, Hussain N, Teng Y (2015) The possible role of chilling in floral and vegetative bud dormancy release in Pyrus pyrifolia. Biol Plant 59:726–734. doi: 10.1007/s10535-015-0547-5Hyndman D, Baumanb DR, Herediac VV, Penning TM (2003) The aldo-keto reductase superfamily homepage. Chem Biol Interact 143–144:621–631. doi: 10.1016/S0009-2797(02)00193-XIto A, Sakamoto D, Moriguchi T (2012) Carbohydrate metabolism and its possible roles in endodormancy transition in Japanese pear. Sci Hortic 144:187–194. doi: 10.1016/j.scienta.2012.07.009Ito A, Sugiura T, Sakamoto D, Moriguchi T (2013) Effects of dormancy progression and low-temperature response on changes in the sorbitol concentration in xylem sap of Japanese pear during winter season. Tree Physiol 33:398–408. doi: 10.1093/treephys/tpt021Jung S, Bassett C, Bielenberg DG et al (2015) A standard nomenclature for gene designation in the Rosaceae. Tree Genet Genomes 11:108. doi: 10.1007/s11295-015-0931-5Kanayama Y, Mori H, Imaseki H, Yamaki S (1992) Nucleotide sequence of a cDNA encoding NADP-sorbitol-6-phosphate dehydrogenase from apple. Plant Physiol 100:1607–1608Kanayama Y, Watanabe M, Moriguchi R, Deguchi M, Kanahama K, Yamaki S (2006) Effects of low temperature and abscisic acid on the expression of the sorbitol-6-phosphate dehydrogenase gene in apple leaves. J Japan Soc Hort Sci 75:20–25. doi: 10.2503/jjshs.75.20Kumar G, Rattan UK, Singh AK (2016a) Chilling-mediated DNA methylation changes during dormancy and its release reveal the importance of epigenetic regulation during winter dormancy in apple (Malus x domestica Borkh). PLoS ONE 11:e0149934. doi: 10.1371/journal.pone.0149934Kumar S, Stecher G, Tamura K (2016b) MEGA7: molecular evolutionary genetics analysis version 70 for bigger datasets. Mol Biol Evol 33:1870–1874. doi: 10.1093/molbev/msw054Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi: 10.1038/227680a0Larkin MA, Blackshields G, Brown NP et al (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948. doi: 10.1093/bioinformatics/btm404Leida C, Terol J, Martí G et al (2010) Identification of genes associated with bud dormancy release in Prunus persica by suppression subtractive hybridization. Tree Physiol 30:655–666. doi: 10.1093/treephys/tpq008Leida C, Conesa A, Llácer G, Badenes ML, Ríos G (2012) Histone modifications and expression of DAM6 gene in peach are modulated during bud dormancy release in a cultivar-dependent manner. New Phytol 193:67–80. doi: 10.1111/j.1469-8137.2011.03863.xLiang D, Cui M, Wu S, Ma F-W (2012) Genomic structure, sub-cellular localization, and promoter analysis of the gene encoding sorbitol-6-phosphate dehydrogenase from apple. Plant Mol Biol Rep 30:904–914. doi: 10.1007/s11105-011-0409-zLiu D, Ni J, Wu R, Teng Y (2013) High temperature alters sorbitol metabolism in Pyrus pyrifolia leaves and fruit flesh during late stages of fruit enlargement. J Am Soc Hortic Sci 138:443–451Lloret A, Conejero A, Leida C et al (2017) Dual regulation of water retention and cell growth by a stress-associated protein (SAP) gene in Prunus. Sci Rep 7:332. doi: 10.1038/s41598-017-00471-7Lo Bianco R, Rieger M, Sung S-JS (2000) Effect of drought on sorbitol and sucrose metabolism in sinks and sources of peach. Physiol Plant 108:71–78. doi: 10.1034/j.1399-3054.2000.108001071.xLoescher WH (1987) Physiology and metabolism of sugar alcohols in higher-plants. Physiol Plant 70:553–557. doi: 10.1111/j.1399-3054.1987.tb02857.xLoescher WH, Marlow GC, Kennedy RA (1982) Sorbitol metabolism and sink-source interconversions in developing apple leaves. Plant Physiol 70:335–339. doi: 10.1104/pp.70.2.335Marquat C, Vandamme M, Gendraud M, Pétel G (1999) Dormancy in vegetative buds of peach: relation between carbohydrate absorption potentials and carbohydrate concentration in the bud during dormancy and its release. Sci Hortic 79:151–162. doi: 10.1016/S0304-4238(98)00203-9Niu Q, Li J, Cai D et al (2016) Dormancy-associated MADS-box genes and microRNAs jointly control dormancy transition in pear (Pyrus pyrifolia white pear group) flower bud. J Exp Bot 67:239–257. doi: 10.1093/jxb/erv454Pfaffl MW, Tichopad A, Prgomet C, Neuvians TP (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—excel-based tool using pair-wise correlations. Biotechnol Lett 26:509–515. doi: 10.1023/B:BILE.0000019559.84305.47Ríos G, Leida C, Conejero A, Badenes ML (2014) Epigenetic regulation of bud dormancy events in perennial plants. Front Plant Sci 5:247. doi: 10.3389/fpls.2014.00247Saito T, Bai S, Imai T, Ito A, Nakajima I, Moriguchi T (2015) Histone modification and signalling cascade of the dormancy-associated MADS-box gene, PpMADS13-1, in Japanese pear (Pyrus pyrifolia) during endodormancy. Plant Cell Environ 38:1157–1166. doi: 10.1111/pce.12469Santamaría ME, Hasbún R, Valera MJ et al (2009) Acetylated H4 histone and genomic DNA methylation patterns during bud set and bud burst in Castanea sativa. J Plant Physiol 166:1360–1369. doi: 10.1016/j.jplph.2009.02.014Shen B, Hohmann S, Jensen RG, Bohnert HJ (1999) Roles of sugar alcohols in osmotic stress adaptation replacement of glycerol by mannitol and sorbitol in yeast. Plant Physiol 121:45–52. doi: 10.1104/pp.121.1.45Sheveleva EV, Marquez S, Chmara W, Zegeer A, Jensen RG, Bohnert HJ (1998) Sorbitol-6-phosphate dehydrogenase expression in transgenic tobacco high amounts of sorbitol lead to necrotic lesions. Plant Physiol 117:831–839. doi: 10.1104/pp.117.3.831Silver N, Best S, Jian J, Thein SL (2006) Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7:33. doi: 10.1186/1471-2199-7-33Talavera G, Castresana J (2007) Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst Biol 56:564–577. doi: 10.1080/10635150701472164Tao R, Uratsu SL, Dandekar AM (1995) Sorbitol synthesis in transgenic tobacco with apple cDNA encoding NADP-dependent sorbitol-6-phosphate dehydrogenase. Plant Cell Physiol 36:525–532. doi: 10.1093/oxfordjournals.pcp.a078789Teo G, Suzuki Y, Uratsu SL et al (2006) Silencing leaf sorbitol synthesis alters long-distance partitioning and apple fruit quality. Proc Natl Acad Sci USA 103:18842–18847. doi: 10.1073/pnas.0605873103Trotel P, Bouchereau A, Niogret MF, Larher F (1996) The fate of osmo-accumulated proline in leaf discs of Rape (Brassica napus L) incubated in a medium of low osmolarity. Plant Sci 118:31–45. doi: 10.1016/0168-9452(96)04422-6Verde I, Abbott AG, Scalabrin S et al (2013) The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nat Genet 45:487–494. doi: 10.1038/ng.2586Webb KL, Burley JWA (1962) Sorbitol translocation in apple. Science 137:766. doi: 10.1126/science.137.3532.766Wisniewski M, Norelli J, Artlip T (2015) Overexpression of a peach CBF gene in apple: a model for understanding the integration of growth, dormancy, and cold hardiness in woody plants. Front Plant Sci 6:85. doi: 10.3389/fpls.2015.00085Yadav R, Prasad R (2014) Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach. Planta 240:223–238. doi: 10.1007/s00425-014-2076-

Transcriptome Analysis of the Vernalization Response in Barley (Hordeum vulgare) Seedlings

Temperate cereals, such as wheat (Triticum spp.) and barley (Hordeum vulgare), respond to prolonged cold by becoming more tolerant of freezing (cold acclimation) and by becoming competent to flower (vernalization). These responses occur concomitantly during winter, but vernalization continues to influence development during spring. Previous studies identified VERNALIZATION1 (VRN1) as a master regulator of the vernalization response in cereals. The extent to which other genes contribute to this process is unclear. In this study the Barley1 Affymetrix chip was used to assay gene expression in barley seedlings during short or prolonged cold treatment. Gene expression was also assayed in the leaves of plants after prolonged cold treatment, in order to identify genes that show lasting responses to prolonged cold, which might contribute to vernalization-induced flowering. Many genes showed altered expression in response to short or prolonged cold treatment, but these responses differed markedly. A limited number of genes showed lasting responses to prolonged cold treatment. These include genes known to be regulated by vernalization, such as VRN1 and ODDSOC2, and also contigs encoding a calcium binding protein, 23-KD jasmonate induced proteins, an RNase S-like protein, a PR17d secretory protein and a serine acetyltransferase. Some contigs that were up-regulated by short term cold also showed lasting changes in expression after prolonged cold treatment. These include COLD REGULATED 14B (COR14B) and the barley homologue of WHEAT COLD SPECIFIC 19 (WSC19), which were expressed at elevated levels after prolonged cold. Conversely, two C-REPEAT BINDING FACTOR (CBF) genes showed reduced expression after prolonged cold. Overall, these data show that a limited number of barley genes exhibit lasting changes in expression after prolonged cold treatment, highlighting the central role of VRN1 in the vernalization response in cereals

Tipping points in the dynamics of speciation.

Speciation can be gradual or sudden and involve few or many genetic changes. Inferring the processes generating such patterns is difficult, and may require consideration of emergent and non-linear properties of speciation, such as when small changes at tipping points have large effects on differentiation. Tipping points involve positive feedback and indirect selection stemming from associations between genomic regions, bi-stability due to effects of initial conditions and evolutionary history, and dependence on modularity of system components. These features are associated with sudden 'regime shifts' in other cellular, ecological, and societal systems. Thus, tools used to understand other complex systems could be fruitfully applied in speciation research

Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat

The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large deletions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these deletions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 promoter region. Twenty-four tetraploid wheat accessions had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley