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

Enhanced AGAMOUS expression in the centre of the Arabidopsis flower causes ectopic expression over its outer expression boundaries

Spatial regulation of C-function genes controlling reproductive organ identity in the centre of the flower can be achieved by adjusting the level of their expression within the genuine central expression domain in Antirrhinum and Petunia. Loss of this control in mutants is revealed by enhanced C-gene expression in the centre and by lateral expansion of the C-domain. In order to test whether the level of central C-gene expression and hence the principle of ‘regulation by tuning’ also applies to spatial regulation of the C-function gene AGAMOUS (AG) in Arabidopsis, we generated transgenic plants with enhanced central AG expression by using stem cell-specific CLAVATA3 (CLV3) regulatory sequences to drive transcription of the AG cDNA. The youngest terminal flowers on inflorescences of CLV3::AG plants displayed homeotic features in their outer whorls indicating ectopic AG expression. Dependence of the homeotic feature on the age of the plant is attributed to the known overall weakening of repressive mechanisms controlling AG. Monitoring AG with an AG-I::GUS reporter construct suggests ectopic AG expression in CLV3::AG flowers when AG in the inflorescence is still repressed, although in terminating inflorescence meristems, AG expression expands to all tissues. Supported by genetic tests, we conclude that upon enhanced central AG expression, the C-domain laterally expands necessitating tuning of the expression level of C-function genes in the wild type. The tuning mechanism in C-gene regulation in Arabidopsis is discussed as a late security switch that ensures wild-type C-domain control when other repressive mechanism starts to fade and fail

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

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

Lineage Conversion of Murine Extraembryonic Trophoblast Stem Cells to Pluripotent Stem Cells▿†

In mammals, the first cell fate decision is initialized by cell polarization at the 8- to 16-cell stage of the preimplantation embryo. At this stage, outside cells adopt a trophectoderm (TE) fate, whereas the inside cell population gives rise to the inner cell mass (ICM). Prior to implantation, transcriptional interaction networks and epigenetic modifications divide the extraembryonic and embryonic fate irrevocably. Here, we report that extraembryonic trophoblast stem cell (TSC) lines are converted to induced pluripotent stem cells (TSC-iPSCs) by overexpressing Oct4, Sox2, Klf4, and cMyc. Methylation studies and gene array analyses indicated that TSC-iPSCs had adopted a pluripotent potential. The rate of conversion was lower than those of somatic reprogramming experiments, probably due to the unique genetic network controlling extraembryonic lineage fixation. Both in vitro and in vivo, TSC-iPSCs differentiated into tissues representing all three embryonic germ layers, indicating that somatic cell fate could be induced. Finally, TSC-iPSCs chimerized the embryo proper and contributed to the germ line of mice, indicating that these cells had acquired full somatic differentiation potential. These results lead to a better understanding of the molecular processes that govern the first lineage decision in mammals