UV-B induced damage and recovery processes in apple leaves as assessed by LIF and PAM fluorescence techniques

The capability of laser-induced chlorophyll fluorescence (LIF) and pulse-amplitude-modulated (PAM) fluorescence technique as well as RED/NIR-light reflection measurements for detection and quantification of UV-B induced damages was evaluated in greenhouse experiments with apple seedlings (Malus domestica Borkh.). Photosynthetic recovery from short-term UV-B stress was assessed during 7 days after UV-B treatment with the PAM fluorometer. The exposure of apple leaves to UV-B doses in the range of 10-26 W m-2 for 180 minutes (UV-BBE dose = 5.4-14 kJ m-2) affected neither chlorophyll content nor leaf reflection. Although UV-B damage was not visually evident 2 hours after irradiation, it could be detected by PAM and LIF fluorescence techniques with equivalent success. The intensity of LIF, estimated as the integral of fluorescence spectrum, was reduced after UV-B irradiation by 19-30%. A stronger decrease in F686 compared to F740 fluorescence resulted in significantly lower F686/F740 values in all UV-B treatments.Apple leaves displayed a strong and significant reduction in maximum fluorescence (Fm) and a slightly increase in ground fluorescence (Fo) 2 hours after UV-B treatment, as documented by PAM fluorescence measurement. Negative linear regressions between investigated UV-B doses and selected PAM parameters were found with determination coefficients (R2) of 0.50 for Fv, 0.48 for Fv/Fm, and 0.58 for Fv/Fo. Among the PAM and LIF parameters tested, the Fv/Fo ratio appeared most sensitive for detection of UV-B induced damages displaying greatest changes and strongest correlation with the applied UV-B doses. PAM fluorescence images of apple leaves visualised an enhanced spatial heterogeneity of photosynthetic activity with increasing UV-B dose. The disturbance in photosynthetic functionality was followed by a continuous recovery process as indicated by restoring Fo and Fm parameters. A decline in maximum photochemical efficiency Fv/Fm from 0.80 to 0.72 and 0.43 after exposure to 20 W m-2 for 240 and 360 minutes (UV-BBE = 14.4 and 21.6 kJ m-2), respectively, was followed by recovery at 7 x 10-4 and 5 x 10-3 units per hour during the first 48 hours after UV-B treatment. The recovery curves of Fm, Fv, Fv/Fm and Fv/Fo parameters during a week after UV-B irradiation were well fitted with exponential rise to maximum function, such as: y = yo + a (1 - e-bx). However, within 7 days after exposure to UV-B light, apple leaves displayed 14% or 4% lower Fm, and 5% or 1% lower Fv/Fm values compared with control plants, indicating only a partial recovery from photoinhibition and irreversible damages in PSII

Novel candidate genes influencing natural variation in potato tuber cold sweetening identified by comparative proteomics and association mapping

BACKGROUND: Higher plants evolved various strategies to adapt to chilling conditions. Among other transcriptional and metabolic responses to cold temperatures plants accumulate a range of solutes including sugars. The accumulation of the reducing sugars glucose and fructose in mature potato tubers during exposure to cold temperatures is referred to as cold induced sweetening (CIS). The molecular basis of CIS in potato tubers is of interest not only in basic research on plant adaptation to environmental stress but also in applied research, since high amounts of reducing sugars affect negatively the quality of processed food products such as potato chips. CIS-tolerance varies considerably among potato cultivars. Our objective was to identify by an unbiased approach genes and cellular processes influencing natural variation of tuber sugar content before and during cold storage in potato cultivars used in breeding programs. We compared by two-dimensional polyacrylamide gel electrophoresis the tuber proteomes of cultivars highly diverse for CIS. DNA polymorphisms in genomic sequences encoding differentially expressed proteins were tested for association with tuber starch content, starch yield and processing quality. RESULTS: Pronounced natural variation of CIS was detected in tubers of a population of 40 tetraploid potato cultivars. Significant differences in protein expression were detected between CIS-tolerant and CIS-sensitive cultivars before the onset as well as during cold storage. Identifiable differential proteins corresponded to protease inhibitors, patatins, heat shock proteins, lipoxygenase, phospholipase A1 and leucine aminopeptidase (Lap). Association mapping based on single nucleotide polymorphisms supported a role of Lap in the natural variation of the quantitative traits tuber starch and sugar content. CONCLUSIONS: The combination of comparative proteomics and association genetics led to the discovery of novel candidate genes for influencing the natural variation of quantitative traits in potato tubers. One such gene was a leucine aminopeptidase not considered so far to play a role in starch sugar interconversion. Novel SNP’s diagnostic for increased tuber starch content, starch yield and chip quality were identified, which are useful for selecting improved potato processing cultivars

Critical function of AP-2gamma/TCFAP2C in mouse embryonic germ cell maintenance

Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E 8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGC-resembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdml activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation

Principles of early human development and germ cell program from conserved model systems

Human primordial germ cells (hPGCs), the precursors of sperm and eggs, originate during week 2-3 of early postimplantation development(1). Using in vitro models of hPGC induction(2-4), recent studies suggest striking mechanistic differences in specification of human and mouse PGCs(5). This may partly be due to the divergence in their pluripotency networks, and early postimplantation development(6-8). Since early human embryos are inaccessible for direct studies, we considered alternatives, including porcine embryos that, as in humans, develop as bilaminar embryonic discs. Here we show that porcine PGCs (pPGCs) originate from the posterior pre-primitive streak competent epiblast by sequential upregulation of SOX17 and BLIMP1 in response to WNT and BMP signalling. Together with human and monkey in vitro models simulating peri-gastrulation development, we show conserved principles for epiblast development for competency for PGC fate, followed by initiation of the epigenetic program(9-11), regulated by a balanced SOX17–BLIMP1 gene dosage. Our combinatorial approach using human, porcine and monkey in vivo and in vitro models, provides synthetic insights on early human development

Identifying fruit characteristics for non-invasive detection of sunburn in apple

Yuri, JA (Antonio Yuri, Jose);Univ Talca, Fac Ciencias Agr, Ctr Pomaceas, Talca, ChileSunburn of apple fruit is one of the major problems in fruit production areas with high light and temperature environment. Fruit with sunburn are predisposed to develop postharvest disorders and need to be separated from the fruit considered for mid to long-term storage. In order to identify specific characteristics for non-invasive, automatic selection of fruit with sunburn, the effect of sunburn on external and internal features on sunlit and shaded fruit sides was assessed in 'Granny Smith', 'Fuji' and 'Braeburn' cultivars. It has been shown that shaded sides of apple with and without sunburn generally do not differ in their internal fruit quality characteristics, Lab colour readings and chlorophyll fluorescence intensities. With sunlit fruit sides, sunburn induced changes in Lab values are cultivar specific. The common feature of fruit with sunburn in all tested cultivars is a greater difference between fruit firmness and soluble solids content on shaded and sunlit sides as compared to unaffected fruit. Due to the chlorophyll breakdown in the affected areas, laser-induced chlorophyll fluorescence (LIF) is proposed as an appropriate sensitive technique for the reliable sunburn detection in green and red apple cultivars. (C) 2011 Elsevier B.V. All rights reserved

Novel in vitro inhibitory functions of potato tuber proteinaceous inhibitors

Plant protease inhibitors are a structurally highly diverse and ubiquitous class of small proteins, which play various roles in plant development and defense against pests and pathogens. Particular isoforms inhibit in vitro proteases and other enzymes that are not their natural substrates, for example proteases that have roles in human diseases. Mature potato tubers are a rich source of several protease inhibitor families. Different cultivars have different inhibitor profiles. With the objective to explore the functional diversity of the natural diversity of potato protease inhibitors, we randomly selected and sequenced 9,600 cDNA clones originated from mature tubers of ten potato cultivars. Among these, 120 unique inhibitor cDNA clones were identified by homology searches. Eighty-eight inhibitors represented novel sequence variants of known plant protease inhibitor families. Most frequent were Kunitz-type inhibitors (KTI), potato protease inhibitors I and II (PIN), pectin methylesterase inhibitors, metallocarboxypeptidase inhibitors and defensins. Twenty-three inhibitors were functionally characterized after heterologous expression in the yeast Pichia pastoris. The purified recombinant proteins were tested for inhibitory activity on trypsin, eleven pharmacological relevant proteases and the non-proteolytic enzyme 5-lipoxygenase. Members of the KTI and PIN families inhibited pig pancreas elastase, beta-Secretase, Cathepsin K, HIV-1 protease and potato 5-lipoxygenase. Our results demonstrate in vitro inhibitory diversity of small potato tuber proteins commonly known as protease inhibitors, which might have biotechnological or medical applications

Derivation and Maintenance of Murine Trophoblast Stem Cells under Defined Conditions

Trophoblast stem cells (TSCs) are in vitro equivalents to the precursor cells of the placenta. TSCs are cultured in serum-rich medium with fibroblast growth factor 4, heparin, and embryonic-fibroblast-conditioned medium. Here, we developed a simple medium consisting of ten chemically defined ingredients for culture of TSCs on Matrigel or synthetic substrates, named TX medium. Gene expression and DNA methylation profiling demonstrated the faithful propagation of expression profiles and epigenomic characteristics of TSCs cultured in TX. Further, TX medium supported the de novo derivation of TSC lines. Finally, TSCs cultured in TX differentiate into all derivatives of the trophectodermal lineage in vitro, give rise to hemorrhagic lesions in nude mice, and chimerize the placenta, indicating that they retained all hallmarks of TSCs. TX media formulation no longer requires fetal bovine serum and conditioned medium, which facilitates and standardizes the culture of this extraembryonic lineage