Role of cAMP in Gibberellin Promotion of Seed Germination in <Emphasis Type="Italic">Orobanche minor</Emphasis> Smith

Adenosine 3′,5′-cyclic monophosphate (cAMP) is known as a key second messenger in many living organisms, regulating a wide range of cellular responses. In higher plants the function of cAMP is poorly understood. In this study, we examined the role of cAMP in seed germination of the root parasitic plant Orobanche minor whose seeds require preincubation in warm moist environments for several days, termed conditioning, prior to exposure to germination stimulants released from roots of host plants. Accumulation of endogenous cAMP was observed in the conditioned O. minor seeds. When the seeds were exposed to light or supraoptimal temperature during the conditioning period, cAMP did not accumulate and the seeds showed low germination rates after stimulation with strigol, a germination stimulant. Addition of membrane-permeable cAMP to the medium restored the germination rates of the seeds treated with light or supraoptimal temperature during the conditioning period, suggesting that cAMP functions during the conditioning period. The endogenous cAMP levels of the seeds conditioned in the light or at a supraoptimal temperature were elevated by treatment of the seeds with gibberellin (GA) during the conditioning period. Uniconazole, a potent inhibitor of GA biosynthesis, blocked elevation of the cAMP level. Furthermore, a correlation between the endogenous cAMP level and GA level was observed during the conditioning period. These results suggest that GAs elevate the cAMP level, which is required for the germination of O. minor seeds.     

Control of macaw palm seed germination by the gibberellin/abscisic acid balance

The hormonal mechanisms involved in palm seed germination are not fully understood. To better understand how germination is regulated in Arecaceae, we used macaw palm (Acrocomia aculeata (Jacq.) Lodd. Ex Mart.) seed as a model. Endogenous hormone concentrations, tocopherol and tocotrienol and lipid peroxidation during germination were studied separately in the embryo and endosperm. Evaluations were performed in dry (D), imbibed (I), germinated (G) and non‐germinated (NG) seeds treated (+GA₃) or not treated (control) with gibberellins (GA). With GA₃ treatment, seeds germinated faster and to a higher percentage than control seeds. The +GA₃ treatment increased total bioactive GA in the embryo during germination relative to the control. Abscisic acid (ABA) concentrations decreased gradually from D to G in both tissues. Embryos of G seeds had a lower ABA content than NG seeds in both treatments. The GA/ABA ratio in the embryo was significantly higher in G than NG seeds. The +GA₃ treatment did not significantly affect the GA/ABA ratio in either treatment. Cytokinin content increased from dry to germinated seeds. Jasmonic acid (JA) increased and 1‐aminocyclopropane‐1‐carboylic acid (ACC) decreased after imbibition. In addition, α‐tocopherol and α‐tocotrienol decreased, while lipid peroxidation increased in the embryo during germination. We conclude that germination in macaw palm seed involves reductions in ABA content and, consequently, increased GA/ABA in the embryo. Furthermore, the imbibition process generates oxidative stress (as observed by changes in vitamin E and MDA).     

Gibberellins in seeds of Arabidopsis thaliana: biological activities,identification and effects of light and chilling on endogenous levels

The activities of several gibberellins in stimulating germination of wild-type and GA-deficient gal seeds of Arabidopsis thaliana were compared. Of the six compounds tested GA₄ and GA₇-isolactone had the highest activity and GA₇ and GA₉ the lowest; activities of GA₁ and GA₃ were intermediate. Combined application of pure GAs presented no indications that more than one GA receptor is involved. Four GAs were identified in extracts from wild-type and GA-insensitive gai seeds by combined gas chromatography mass spectrometry: GA₁, GA₃, GA₄ and GA₉. Effects of light and chilling on levels of GA₁, GA₄ and GA₉ were studied using deuterated standards. Light increased both GA levels and germination in unchilled wild-type and gai seeds. As a result of irradiation GA levels in gai seeds were 7–10 times as high as in wild-type seeds. In the dark germination was 0%, in the light 14% of gai seeds and 95% of wild-type seeds germinated. A chilling pre-treatment of 7 days at 2°C was required to enhance further the germination of gai seeds in the light. Light did not increase GA levels of chilled seeds of either genotype; levels of GA₄ and GA₉ of chilled gai seeds, in the light were respectively 7 and 12 times lower than in non-chilled seeds, whereas the latter seeds germinated better. Slightly elevated levels of GA₄ were detected in darkness after chilling, but germination capacity was still 0%. These results strengthened the conclusion that GAs are required for germination of A. thaliana seeds, whereby GA₄ has intrinsic biological activity. However, it is unlikely that light and chilling stimulate germination primarily by increasing levels of GA. Instead GA sensitivity is a possible alternative.     

Cytochrome <Emphasis Type="Italic">c</Emphasis>-induced lymphocyte death from the outside in: inhibition by serum leucine-rich alpha-2-glycoprotein-1

Previously we reported that serum leucine-rich alpha-2-glycoprotein-1 (LRG) binds cytochrome c (Cyt c; Cummings et al., Apoptosis 11:1121–1129, 2009). Here we show that LRG binding to Cyt c is similar to that of Apaf-1. LRG and Apaf-1 share partial amino acid sequences, compete for binding Cyt c, and are inhibited by modification at lysine 72 in Cyt c. However, in contrast to Apaf-1, LRG acts as a survival factor in vitro rather than a pro-apoptotic factor. By depleting LRG from culture medium we found that LRG protects against a toxic effect of exogenous Cyt c on lymphocytes that would otherwise result in an apoptotic phenotype. LRG, as well as antibodies specific for Cyt c, increased cell viability in the absence of added Cyt c indicating that Cyt c released by dying cells in the cultures is itself toxic. Protection from extracellular Cyt c-induced lymphotoxicity appears to involve an active mechanism rather than steric hindrance of Cyt c. Thus, serum LRG when bound to extracellular Cyt c that is released from apoptotic cells acts as a survival factor for lymphocytes and possibly other cells that are susceptible to the toxic effect of extracellular Cyt c.     

Disrupting Abscisic Acid Homeostasis in Western White Pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) Seeds Induces Dormancy Termination and Changes in Abscisic Acid Catabolites

To investigate the role of abscisic acid (ABA) biosynthesis and catabolism in dormant imbibed seeds of western white pine (Pinus monticola), ABA and selected catabolites were measured during a combined treatment of the ABA biosynthesis inhibitor fluridone, and gibberellic acid (GA). Fluridone in combination with GA effectively disrupted ABA homeostasis and replaced the approximately 90-day moist chilling period normally required to break dormancy in this species. Individually, both fluridone and GA treatments decreased ABA levels in the embryos and megagametophytes of white pine seeds compared to a water control; however, combined fluridone/GA treatment, the only treatment to terminate dormancy effectively, led to the greatest decline in ABA content. Fluridone treatments revealed that a high degree of ABA turnover/transport occurred in western white pine seeds during the initial stages of dormancy maintenance; at this time, ABA levels decreased by approximately two-thirds in both embryo and megagametophyte tissues. Gibberellic acid treatments, both alone and in combination with fluridone, suggested that GA acted transiently to disrupt ABA homeostasis by shifting the ratio between biosynthesis and catabolism to favor ABA catabolism or transport. Increases in phaseic acid (PA) and dihydrophaseic acid (DPA) were observed during fluridone/GA treatments; however, increases in ABA metabolites did not account for the reduction in ABA observed; additional catabolism and/or transport of ABA and selected metabolites in all probability accounts for this discrepancy. Finally, levels of 7′ hydroxy-ABA (7′OH-ABA) were higher in dormant-imbibed seeds, suggesting that metabolism through this pathway is increased in seeds that maintain higher levels of ABA, perhaps as a means to further regulate ABA homeostasis.     

Selection and characterization of a rice mutant resistant to 5-methyltryptophan

Summary A rice plant resistant to 5-methyltryptophan (5MT) was selected from mutagenized M3 seeds (Oryza sativa L. var. Sasanishiki) originating from panicles treated with ethylene imine (0.2%) 2 h after flowering. When germinated on 5MT-containing medium, the seeds (M4) from selfed plants segregated with a 3 resistant:1 sensitive ratio, indicating that the plant was heterozygous for a resistance gene and that the resistance was dominant. The resistance was also expressed in callus derived from seeds. Analysis of the free amino acids in seeds, seedlings, and calli showed that homozygous resistant plants (TR1) contained higher levels of total free amino acids than sensitive plants. In particular the levels of tryptophan, phenylalanine, and histidine were, respectively, 8.5, 5.4, and 4.9 times higher than those in the sensitive plants.     

Early Sprouting and First Stages of Growth of Rice Seeds Exposed to a Magnetic Field

The effects of a stationary magnetic field on the germination of rice seeds (Oryza sativa, L.) and on the initial stages of growth of rice plants have been evaluated. In both tests, the seeds were exposed to one of two magnetic field strengths (125 or 250 mT) for different times (1 min, 10 min, 20 min, 1 h, 24 h, or chronic exposure) as 12 separate treatments (doses D1–D6 for 125 mT and doses D7–D12 for 250 mT). Nonexposed seeds were used as controls (C). The mean germination time (MGT) was significantly reduced compared to control when seeds were exposed to magnetic field (54.00 h for D5 and D11, and 58.56 h for control). The parameters T₁ and T₁₀, times required to obtain 1 and 10% of germinated seeds, were also reduced. The T₁₀ of control seeds was 44.40 h, while treatments D5, D6, D11, and D12 gave rise to values of 36.00, 36.96, 32.64, and 39.36 h, respectively. The higher germination rate of treated seeds obtained in the germination test is in agreement with the higher lengths and weights of rice plants exposed to a magnetic field recorded on the growth tests. All the parameters measured were over the control ones, although the highest lengths and weights of rice plants were obtained for chronic exposure to magnetic field (doses D6 and D12). Stem length of control plants (45.36 mm) measured at the tenth day was significantly lower than that obtained for doses D6 (58.58 mm) and D12 (80.63 mm); the same behavior was observed on total length, stem weight, and total weight. Our finding indicates that this type of magnetic treatment clearly affects germination and the first stages of growth of rice plants.     

Gibberellin content of immature apple seeds from paclobutrazol-treated trees over three seasons

Seeds from heavily fruiting (on-year), mature untreated, and paclobutrazol-treated apple trees (Malus domestica Borkh. cv. Spartan) were sampled in mid-June 1987, mid-July 1987, and mid-July 1990. After seeds were freeze-dried, gibberellins (GAs) were extracted, purified, and fractionated via C₁₈ reversed-phase high-performance liquid chromatography (HPLC). Nine GAs (GA₁, GA₃, GA₄, GA₇, GA₈, GA₉, GA₁₉, GA₂₀, and GA₅₃) were quantified by the use of deuterated GA internal standards. Paclobutrazol trunk drench treatments reduced vegetative shoot elongation in the seasons that seeds were sampled by 55% or more. Between June 17, 1987 and July 15, 1987, the dry weight of seeds from both untreated and treated trees increased about 2.5 times and there were reductions, on a per seed basis, of GA₄ in seeds from both untreated and treated trees, of GA₇ in seeds from treated trees, and of GA₉ in seeds from untreated trees. However, GA₉ increased in seeds from treated trees. Changes in levels of some of the early-13-hydroxylation pathway GAs (GA₁₅ GA₃, GA₈, GA₁₉, GA₂₀, and GA₅₃) also occurred during the month. For mid-July harvested seeds, the pattern, with some exceptions, was that 2 years after paclobutrazol treatment (1987), levels of early-13-hydroxylation pathway GAs in seeds from treated trees were lower compared to controls but after 5 years (1990) their levels tended to increase. For the non-13-hydroxylated GAs (GA₄, GA₇, and GA₉), 2 years after paclobutrazol treatment, GA₄ levels were equal in seeds from untreated and treated trees, GA₇ decreased in seeds from treated trees compared with controls, but GA₉ levels increased. Levels of these three GAs were higher in seeds from treated trees 5 years after treatment (1990) but levels of GA₄, GA₇, and GA₉ dramatically increased in seeds from treated trees 4 years after treatment (1989), as we previously reported.     

Gibberellin Metabolism and Transport During Germination and Young Seedling Growth of Pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The role of gibberellins (GAs) during germination and early seedling growth is examined by following the metabolism and transport of radiolabeled GAs in cotyledon, shoot, and root tissues of pea (Pisum sativum L.) using an aseptic culture system. Mature pea seeds have significant endogenous GA₂₀ levels that fall during germination and early seedling growth, a period when the seedling develops the capacity to transport GA₂₀ from the cotyledon to the shoot and root of the seedling. Even though cotyledons at 0–2 days after imbibition have appreciable amounts of GA₂₀, the cotyledons retain the ability to metabolize labeled GA₁₉ to GA₂₀ and express significant levels of PsGA20ox2 message (which encodes a GA biosynthesis enzyme, GA 20-oxidase). The large pool of cotyledonary GA₂₀ likely provides substrate for GA₁ synthesis in the cotyledons during germination, as well as for shoots and roots during early seedling growth. The shoots and roots express GA metabolism genes (PsGA3ox genes which encode GA 3-oxidases for synthesis of bioactive GA₁, and PsGA2ox genes which encode GA 2-oxidases for deactivation of GAs to GA₂₉ and GA₈), and they develop the capacity to metabolize GAs as necessary for seedling establishment. Auxins also show an interesting pattern during early seedling growth, with higher levels of 4-chloro-indole-3-acetic acid (4-Cl-IAA) in mature seeds and higher levels of indole-3-acetic acid (IAA) in young root and shoot tissues. This suggests a changing role for auxins during early seedling development.     

AtPER1 enhances primary seed dormancy and reduces seed germination by suppressing the ABA catabolism and GA biosynthesis in Arabidopsis seeds

Seed is vital to the conservation of germplasm and plant biodiversity. Seed dormancy is an adaptive trait in numerous seed‐plant species, enabling plants to survive under stressful conditions. Seed dormancy is mainly controlled by abscisic acid (ABA) and gibberellin (GA) and can be classified as primary and secondary seed dormancy. The primary seed dormancy is induced by maternal ABA. Here we found that AtPER1, a seed‐specific peroxiredoxin, is involved in enhancing primary seed dormancy. Two loss‐of‐function atper1 mutants, atper1‐1 and atper1‐2, displayed suppressed primary seed dormancy accompanied with reduced ABA and increased GA contents in seeds. Furthermore, atper1 mutant seeds were insensitive to abiotic stresses during seed germination. The expression of several ABA catabolism genes (CYP707A1, CYP707A2, and CYP707A3) and GA biosynthesis genes (GA20ox1, GA20ox3, and KAO3) in atper1 mutant seeds was increased compared to wild‐type seeds. The suppressed primary seed dormancy of atper1‐1 was completely reduced by deletion of CYP707A genes. Furthermore, loss‐of‐function of AtPER1 cannot enhance the seed germination ratio of aba2‐1 or ga1‐t, suggesting that AtPER1‐enhanced primary seed dormancy is dependent on ABA and GA. Additionally, the level of reactive oxygen species (ROS) in atper1 mutant seeds was significantly higher than that in wild‐type seeds. Taken together, our results demonstrate that AtPER1 eliminates ROS to suppress ABA catabolism and GA biosynthesis, and thus improves the primary seed dormancy and make the seeds less sensitive to adverse environmental conditions.     

Characterization of highly variable (GA/CT) n microsatellites in the bur oak,Quercus macrocarpa

The objective of this study was to ascertain the usefulness of polymerase chain reaction (PCR)-based microsatellite analysis for studying pollination and parentage in a wind-pollinated temperate tree. A small insert genomic library of the bur oak (Quercus macrocarpa) was constructed and screened for the presence of (CA/GT) _n and (GA/CT) _n repeats. The proportion of positive clones yielded estimates of 3×10⁵ such dinucleotide repeats per genome, roughly comparable to abundances reported in other eukaryotic genomes. Thirteen positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT) _n motif was more abundant than the (CA/GT) _n motif in these clones. The (GA/CT) _n repeats also showed longer average repeat length (mean n=16.2 versus 7.3), suggesting that they are better candidates for yielding polymorphic genetic markers in oak genomes. Indeed, a survey of adult bur oaks and offspring in a small stand in northern Illinois at 3 of these (GA/CT) _n microsatellite loci revealed Mendelian inheritance and extremely high levels of polymorphism, with the number of alleles at each locus ranging from 11–20 and heterozygosity ranging from 0.66 to 0.75. These results, indicating that (GA/CT) _n microsatellites are both abundant and highly polymorphic in the bur oak genome, suggest that such genetic markers have tremendous potential for applications for studies of parentage, pollination and dispersal in temperate trees.     

The effect of stratification temperatures on the level of dormancy in primary and secondary dormant seeds of two <Emphasis Type="Italic">Carex</Emphasis> species

The effect of temperature on the level of dormancy of primary and secondary dormant Carex pendula and Carex remota seeds was investigated. Primary dormant and secondary dormant seeds were stratified for 4 weeks at 5, 11, 13, and 15 °C, respectively, and tested for germination at 15/5 °C in light. To obtain secondary dormant seeds, primary dormant seeds were stratified at 5 °C and afterwards at 25 °C for 4 weeks. Germination tests were carried out in water and in 25 μmol KNO₃-solution to examine differences in sensitivity to nitrate between seeds relieved from primary and secondary dormancy. In both species, seeds with primary and with induced secondary dormancy showed no significant differences in germination. The two sedges showed significant differences in the effect of stratification temperatures between primary and secondary dormant seeds. Primary dormant seeds of C. pendula showed high germination (>80%) in nitrate-solution after stratification at all temperatures, while only temperatures of 5, 11, and 13 °C led to higher germination in nitrate-solution in secondary dormant seeds. Germination percentages of primary and of secondary dormant C. pendula seeds in water increased to a higher extent only after stratification at 5 and 11 °C; stratification of 11 °C was more effective in secondary than in primary dormant seeds. The only temperature that relieved primary dormancy in C. remota seeds was 5 °C where germination in water and nitrate-solution was >90%. Germination of secondary dormant seeds was increased by stratification at 11 °C independent of the test solution but higher germination after stratification at 13 °C occurred only in nitrate-solution. The results support the existence of physiological differences in the regulation of primary and secondary dormancy by temperature, and in the reaction of nitrate, at least in C. remota.     

Size and number of tandem repeat arrays can determine somatic homologous pairing of transgene loci mediated by epigenetic modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> nuclei

The chromosomal arrangement of different transgenic repeat arrays inserted at various chromosomal positions was tested by FISH in Arabidopsis 2C leaf and root nuclei. Large lacO (∼10 kb) but not tetO (4.8 kb) or small lacO (∼2 kb) arrays were, in general, more often spatially associated with heterochromatic chromocenters (CC) than flanking regions (that either overlap the array insert position or are between 5 and 163 kb apart from the insert site). Allelic and ectopic pairing frequencies of lacO arrays were significantly increased only in nuclei of lines with two large lacO arrays inserted at different positions on the same chromosome arm. Within the same lines, root nuclei showed a significantly lower increase of pairing frequencies at the insert position compared to leaf nuclei but still a higher frequency than in the wild-type situation. Thus, the frequencies of homologous pairing and association with heterochromatin of transgenic repeats may differ with the construct, the chromosomal insertion position, the cell type and with the number and repetitiveness of inserts. Strong CpG methylation is correlated with a high frequency of homologous pairing at large repeat array loci in somatic cells but has no impact on their association with CCs. These results show that single low-copy arrays apparently do not alter interphase chromatin architecture and are more suitable for chromatin tagging than multiple high copy arrays.     

Validation of LRG1 as a Potential Biomarker for Detection of Epithelial Ovarian Cancer by a Blinded Study

Background

Leucine-rich alpha-2-glycoprotein (LRG1) was found to be differentially expressed in sera from patients with Epithelial Ovarian Cancer (EOC). The aim of this study is to investigate the performance of LRG1 for detection of EOC, including early stage EOC, and to evaluate if LRG1 can complement CA125 in order to improve EOC detection using two independent blinded sample sets.

Methods and Results

Serum LRG1 and CA125 were measured by immunoassays. All assays were performed blinded to clinical data. Using the two independent sample sets (156 participants for sample set 1, and 233 for sample set 2), LRG1 was differentially expressed in EOC cases as compared to healthy, surgical, and benign controls, and its performance was not affected by the conditions of blood collection. The areas under the ROC curve (AUC) for LRG1 in differentiating EOC cases from non-cases were 0.797 and 0.786 for sample set 1 and 2. For differentiating EOC cases from healthy controls, the AUC values for LRG1 were 0.792 and 0.794. At a fixed specificity of 95%, LRG1 detects 52%, and 53.5% of EOC cases from healthy controls for sample set 1 and 2. When combining LRG1 and CA125, the AUC value increased to 0.927, which was improved compared to CA125 (AUC=0.916) (p=0.008) alone in distinguishing EOC cases from non-cases. More importantly, LRG1 also showed potential performance in differentiating early stage EOC from non-cases with an AUC of 0.715 for sample set 1, and 0.690 for sample set 2. The combination of LRG1 and CA125 resulted in an AUC of 0.838, which outperforms CA125 (AUC=0.785) (p=0.018) in detecting early stage EOC cases from non-cases using the larger sample set.

Conclusions

LRG1 could be a useful biomarker alone or in combination with CA125 for the diagnosis of ovarian cancer.