The distribution of ATP within tomato (Lycopersicon esculentum Mill.) embryos correlates with germination whee as total ATP concentration does not

The distribution of ATP in tomato seeds was visualized by monitoring the luminescence of frozen sections on top of a gel containing all the components of the luciferase reaction, but excluding ATP. ATP was imaged in germinating tomato seeds at intervals of 3, 6, 17, 24 and 48 h and in seeds with primary or secondary dormancy. ATP was present mainly in the embryo and concentrated in the radicle tip towards the completion of germination. In contrast to germinating seeds, ATP was distributed more evenly in dormant seeds. For germination, the ratio of ATP concentration in the radicle tip versus cotyledons was decisive, rather than the absolute concentration

Seed dormancy and germination : light and nitrate

One of the most important aspects of the life cycle of seed plants is the formation and development of seeds on the motherplant and the subsequent dispersal. An equally important element of the survival strategy is the ability of seeds to prevent germination in unfavorable conditions, such as the wrong season, low light irradiance, or an unfavorable soil composition. Seeds of many species may remain in the soil, either in a dry state or fully imbibed, for hundreds of years without losing their viability. Earlier investigations have shown that this state of dormancy may be broken and induced in a seasonal cycle. Evidently, temperature is an important regulating factor in these cycles. When dormancy is broken, seeds may germinate, providing the conditions are adequate. Hence, seeds must be able to 'sense' their environment. Again, temperature is important, but also two naturally occurring environmental factors, light and nitrate, are known to stimulate the germination of many (wild) species.Seeds of Sisymbrium officinale (hedge mustard) only germinate in the presence of light and nitrate (either endogenous or exogenous). Irradiation with far-red light (720 run) antagonized the stimulating action of red light (660 nm). Both wavelengths are part of the daylight spectrum. This is proof that the light-induced germination is mediated by the plant pigment phytochrome. The induction of germination by light and nitrate could also be inhibited by application of tetcyclacis, an inhibitor of the biosynthesis of gibberellins. Application of gibberellins 4 and 7 antagonized the inhibition. It was concluded that synthesis of gibberellins is part of the transduction chain that leads to germination. Comparison of the escape times for the antagonizing action of far-red light and the inhibition of tetcyclacis led to the conclusion that induction of germination occurred during the first 8 hours after irradiation with red light and application of nitrate, while the synthesis of gibberellins was completed for all seeds after 16 hours.Seeds only germinated in the dark when exogenous gibberellins were present. In the absence of nitrate, red light reduced the requirement for gibberellins. In other words, besides a promotive effect on gibberellin synthesis (light-effect I), light also enhanced the sensitivity to gibberellins (light-effect II). Light-effect I disappeared after prolonged treatment at elevated temperatures while the seeds remained responsive to exogenous gibberellins and light-effect II (Chapter 2).Both light-effects were studied in detail (Chapter 3). The influence of several concentrations of nitrate and gibberellins on the shape and position of fluence-response curves was examined. The results obtained with seeds of Sisymbrium officinale were compared with those of the gibberellin-deficient mutant ofArabidopsis thaliana, a related species. Remarkable similarities were observed. In both cases nitrate steepened the fluence-response curves. In other words, the fluence-range over which the total seed population responded was reduced. Application of gibberellins 4 and 7 to seeds, in the absence of exogenous nitrate, resulted for both species in a parallel shift to lower fluence values. Application of cofactor analysis led to the conclusion that the interaction between the effects of nitrate and phytochrome was multiplicative. The germination response was a function of the product of nitrate concentration and fluence value. This is an indication that the factors nitrate and phytochrome act on the same pathway. The interaction between the effects of phytochrome and gibberellins, however, was additive. Both factors acted independently in different pathways leading to the same response. Phytochrome might enhance the sensitivity of the gibberellin- receptors (light-effect II). However, this effect could only be brought to expression when sufficient gibberellins were present. The use of the gibberellindeficientArabidopsis mutantsupported the hypothesis that in Sisymbrium no active gibberellins are present in the absence of nitrate.The hypothesis that nitrate is active in the induction of germination because its reduction would lead to production of NADP, stimulator of the pentose phosphate pathway, was tested by studying the light- and nitrateinduced germination in the presence of inhibitors of nitrate reductase (Chapter 4). Furthermore, a method was developed to measure the nitrate content of seeds. The inhibitors of nitrate reductase, sodium chlorate and sodium tungstate had no influence on the light-induced germination in a range of nitrate concentrations. At several intervals after a pretreatment during which the seeds had taken up nitrate from the medium, the nitrate contents of the seeds and the medium were measured. It was found that during the induction of germination, the 8-hour period after irradiation, the nitrate content of the seeds decreased. However, this decrease could be fully explained by leakage into the medium. Thus, no nitrate was reduced during this period. Nitrate reduction did not occur until actual growth (protrusion of the radicle) had started. Apparently, at that time the nitrate assimilation began. In the presence of the inhibitors this reduction was almost completely inhibited. The growth of the seedling was abnormally slow in the presence of the inhibitors. These results led to the conclusion that nitrate is active in the unreduced state at induction of germination.To gain more insight in the process of dormancy induction and the decreasing sensitivity to light and nitrate during this process, doseresponse experiments were carried out for these factors during induction of dormancy at constant temperature (Chapter 5). Fluence - response curves of seeds in supra-optimal nitrate concentrations shifted to higher fluence values upon increasing duration of the pretreatment. After approximately 120 hours at 15°C the maximum germination decreased. The slopes of the curves did not change. The observed fluence-response curves could be simulated by formulations from the general receptor -occupancy theory. This enabled us to interpret calculated curve parameters as interaction parameters of the binding of phytochrome to its receptor. The shifts of the curves with unchanged maximal response could in this way be explained by assuming that more phytochrome -receptors were present than required for maximal germination. The induction of secondary dormancy could then be the result of a temperature dependent decrease of the number of receptors. Once below a critical value this decrease could lead to reduction of the maximum response.The role of nitrate was studied in a similar way (Chapter 6). The response to nitrate was monitored in optimal light conditions. The results of the nitrate-response experiments showed similarities with those of the fluence-response experiments. Upon increasing pretreatment duration a shift to higher nitrate concentrations was observed, followed by a decrease of the maximum germination. The nitrate-response curves could also be simulated with an equation for a simple bimolecular reaction. Furthermore, it was shown that the presence of nitrate was an absolute requirement for the light-induced germination. It was concluded that nitrate may act as an activator of the phytochrome-receptors. In the absence of exogenous nitrate, the response was limited by the amount of endogenous nitrate. Since nitrate leached out during incubation in water the germination in water was correlated with the nitrate leaching. A remarkable aspect of the nitrate -responses was the occurrence of biphasic response curves. The very low nitrate response disappeared after 48-72 hours of pretreatment. The occurrence of these two phases was related to the uptake of nitrate from a range of nitrate concentrations. For the first phase the uptake appeared to be the limiting factor; all the nitrate that was taken up was bound. For the second phase relatively high endogenous nitrate levels were required. A considerable amount of this nitrate was not bound. The possibility of the existence of two different nitrate receptors was discarded. By analogy with an existing model for biphasic fluence-response curves it was suggested that the requirement of the nitrate -receptor for nitrate for a certain fraction of the seed population could differ from the rest of the population. Seemingly, induction of dormancy could influence the ratio of these fractions.The results were summarized in a model in which the phytochrome-receptor is located in a membrane (Chapter 7). Breakage of dormancy, regulated by temperature, would induce synthesis of this receptor. Changing the temperature to an optimal germination temperature would induce a phase transition of the membrane, thus enabling the receptor to move laterally in the membrane. In this way the receptor may become accessible to nitrate. Nitrate would bind to the receptor and alter its conformation in such a way that phytochrome can bind. The transduction chain, leading to germination, may be initiated via a secondary messenger like calcium. Induction of dormancy makes this process impossible because the rate of degradation of phytochrome-receptors will exceed the synthesis.</TT

Seed vigor, aging, and osmopriming affect anion and sugar leakage during imbition of maize (Zea mays L.) caryopses

Conductivity was significantly increased by aging and decreased by osmopriming of maize (Zea mays L.) caryopses. Chloride, phosphate, and sulfate were the main anions that leaked out of maize seeds; their leakage was closely related to conductivity, increased by aging, and decreased by osmopriming. The anion leakage of isolated embryos correlated closely to seed vigor and was more sensitive to aging and priming than that of the whole seed. Anion leakage may be a more sensitive measure for seed vigor than bulk conductivity readings. Aging did not increase the sugar leakage of whole seeds but significantly increased the sugar leakage of isolated embryos. Sugar leakage was not closely related to total soluble sugar content of seeds. While priming decreased seed conductivity, the decreased anion and sugar leakage of the primed seeds was mainly caused by the washing effect during priming. The total anions or sugars left in the polyethylene glycol (PEG) solution after priming and in the conductivity solution of the primed seeds was almost the same as in the conductivity solution of the unprimed seeds alone

Advances in Genetical Genomics of Plants

Natural variation provides a valuable resource to study the genetic regulation of quantitative traits. In quantitative trait locus (QTL) analyses this variation, captured in segregating mapping populations, is used to identify the genomic regions affecting these traits. The identification of the causal genes underlying QTLs is a major challenge for which the detection of gene expression differences is of major importance. By combining genetics with large scale expression profiling (i.e. genetical genomics), resulting in expression QTLs (eQTLs), great progress can be made in connecting phenotypic variation to genotypic diversity. In this review we discuss examples from human, mouse, Drosophila, yeast and plant research to illustrate the advances in genetical genomics, with a focus on understanding the regulatory mechanisms underlying natural variation. With their tolerance to inbreeding, short generation time and ease to generate large families, plants are ideal subjects to test new concepts in genetics. The comprehensive resources which are available for Arabidopsis make it a favorite model plant but genetical genomics also found its way to important crop species like rice, barley and wheat. We discuss eQTL profiling with respect to cis and trans regulation and show how combined studies with other ‘omics’ technologies, such as metabolomics and proteomics may further augment current information on transcriptional, translational and metabolomic signaling pathways and enable reconstruction of detailed regulatory networks. The fast developments in the ‘omics’ area will offer great potential for genetical genomics to elucidate the genotype-phenotype relationships for both fundamental and applied research

A gene co-expression network predicts functional genes controlling the re-establishment of desiccation tolerance in germinated Arabidopsis thaliana seeds.

MAIN CONCLUSION: During re-establishment of desiccation tolerance (DT), early events promote initial protection and growth arrest, while late events promote stress adaptation and contribute to survival in the dry state. Mature seeds of Arabidopsis thaliana are desiccation tolerant, but they lose desiccation tolerance (DT) while progressing to germination. Yet, there is a small developmental window during which DT can be rescued by treatment with abscisic acid (ABA). To gain temporal resolution and identify relevant genes in this process, data from a time series of microarrays were used to build a gene co-expression network. The network has two regions, namely early response (ER) and late response (LR). Genes in the ER region are related to biological processes, such as dormancy, acquisition of DT and drought, amplification of signals, growth arrest and induction of protection mechanisms (such as LEA proteins). Genes in the LR region lead to inhibition of photosynthesis and primary metabolism, promote adaptation to stress conditions and contribute to seed longevity. Phenotyping of 12 hubs in relation to re-establishment of DT with T-DNA insertion lines indicated a significant increase in the ability to re-establish DT compared with the wild-type in the lines cbsx4, at3g53040 and at4g25580, suggesting the operation of redundant and compensatory mechanisms. Moreover, we show that re-establishment of DT by polyethylene glycol and ABA occurs through partially overlapping mechanisms. Our data confirm that co-expression network analysis is a valid approach to examine data from time series of transcriptome analysis, as it provides promising insights into biologically relevant relations that help to generate new information about the roles of certain genes for DT

Depth of dormancy in tomato (Lycopersicon esculentum Mill.) seeds is related to the progression of the cell cycle prior to the induction of dormancy

Cell cycle activities are initiated following imbibition of non-dormant seeds. However, it is not known whether cell cycle related events other than DNA replication also remain suppressed in imbibed dormant seeds. The objective of this study was to demonstrate that the transitions between the non-dormant and dormant (both primary and secondary) states are reflected in cell cycle events, such as DNA replication and the changing patterns of the microtubular cytoskeleton involved in the processes of growth and development. The present studies were conducted on seeds from tomato (Lycopersicon esculentum cv. Moneymaker) that possessed primary dormancy or were manipulated to attain secondary dormancy. In addition, a non-dormant abscisic acid (ABA)-deficient mutant, sitw, was used. DNA replication, as measured by flow cytometry, and -tubulin accumulation, analysed by immunoblotting, were compared with immunocytological studies of active DNA synthesis and microtubular cytoskeleton formation. It is shown that the depth of dormancy, which distinguishes primary and secondary dormancy, may depend on the progression of the cell cycle prior to the induction of dormancy

Exploring the Natural Variation for Seedling Traits and Their link with Seed Dimensions in Tomato

The success of germination, growth and final yield of every crop depends to a large extent on the quality of the seeds used to grow the crop. Seed quality is defined as the viability and vigor attribute of a seed that enables the emergence and establishment of normal seedlings under a wide range of environments. We attempt to dissect the mechanisms involved in the acquisition of seed quality, through a combined approach of physiology and genetics. To achieve this goal we explored the genetic variation found in a RIL population of Solanum lycopersicum (cv. Moneymaker) x Solanum pimpinellifolium through extensive phenotyping of seed and seedling traits under both normal and nutrient stress conditions and root system architecture (RSA) traits under optimal conditions. We have identified 62 major QTLs on 21 different positions for seed, seedling and RSA traits in this population. We identified QTLs that were common across both conditions, as well as specific to stress conditions. Most of the QTLs identified for seedling traits co-located with seed size and seed weight QTLs and the positive alleles were mostly contributed by the S. lycopersicum parent. Co-location of QTLs for different traits might suggest that the same locus has pleiotropic effects on multiple traits due to a common mechanistic basis. We show that seed weight has a strong effect on seedling vigor and these results are of great importance for the isolation of the corresponding genes and elucidation of the underlying mechanisms

Triple-negative breast cancer: Present challenges and new perspectives

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