Temperature fine-tunes Mediterranean Arabidopsis thaliana life-cycle phenology geographically.

To understand how adaptive evolution in life-cycle phenology operates in plants, we need to unravel the effects of geographic variation in putative agents of natural selection on life-cycle phenology by considering all key developmental transitions and their co-variation patterns. We address this goal by quantifying the temperature-driven and geographically varying relationship between seed dormancy and flowering time in the annual Arabidopsis thaliana across the Iberian Peninsula. We used data on genetic variation in two major life-cycle traits, seed dormancy (DSDS50) and flowering time (FT), in a collection of 300 A. thaliana accessions from the Iberian Peninsula. The geographically varying relationship between life-cycle traits and minimum temperature, a major driver of variation in DSDS50 and FT, was explored with geographically weighted regressions (GWR). The environmentally varying correlation between DSDS50 and FT was analysed by means of sliding window analysis across a minimum temperature gradient. Maximum local adjustments between minimum temperature and life-cycle traits were obtained in the southwest Iberian Peninsula, an area with the highest minimum temperatures. In contrast, in off-southwest locations, the effects of minimum temperature on DSDS50 were rather constant across the region, whereas those of minimum temperature on FT were more variable, with peaks of strong local adjustments of GWR models in central and northwest Spain. Sliding window analysis identified a minimum temperature turning point in the relationship between DSDS50 and FT around a minimum temperature of 7.2 °C. Above this minimum temperature turning point, the variation in the FT/DSDS50 ratio became rapidly constrained and the negative correlation between FT and DSDS50 did not increase any further with increasing minimum temperatures. The southwest Iberian Peninsula emerges as an area where variation in life-cycle phenology appears to be restricted by the duration and severity of the hot summer drought. The temperature-driven varying relationship between DSDS50 and FT detected environmental boundaries for the co-evolution between FT and DSDS50 in A. thaliana. In the context of global warming, we conclude that A. thaliana phenology from the southwest Iberian Peninsula, determined by early flowering and deep seed dormancy, might become the most common life-cycle phenotype for this annual plant in the region.

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.

Dose-Response Analysis of Factors Involved in Germination and Secondary Dormancy of Seeds of Sisymbrium officinale: I. Phytochrome.

The germination of seeds of Sisymbrium officinale is light- and nitrate dependent. A close interaction between the effects of light and nitrate on germination has been reported previously (HWM Hilhorst, CM Karssen [1988] Plant Physiol 86: 591-597). In this study, a detailed dose-response analysis of the light-induced germination during induction of secondary dormancy is presented. Germination in water dropped from 90 to 0% after a dark incubation of 15 degrees C of approximately 160 hours. In the presence of 25 millimolar KNO(3), the decrease in germination level was delayed. At 24-hour intervals fluence-response curves were obtained in the presence of 25 millimolar KNO(3). With increasing length of the preincubation period, fluence-response curves shifted along the abscissa to the right. After 120 hours the maximal germination level started to decline. The fluence-response curves were simulated by using formulations from receptor occupancy theory for a simple bimolecular reaction in which the reaction partners were Pfr and its tentative receptor X. A good simulation was obtained when cooperativity of the binding of Pfr to X was assumed. The experimental curve parameters could then be interpreted as binding parameters.

Dose-Response Analysis of Factors Involved in Germination and Secondary Dormancy of Seeds of Sisymbrium officinale: II. Nitrate.

The role of nitrate as a promoter of germination of Sisymbrium officinale seeds was examined in optimal light conditions. It was shown that the requirement for nitrate was absolute. This was true for all seed lots used. The probit of germination in water was log-linearly related to the level of endogenous nitrate. Preincubation at 15 degrees C resulted in an immediate decrease in germination, whereas in 25 millimolar KNO(3) the decrease was delayed. The decline of germination in water was strongly correlated with the rate at which nitrate leached from the seeds. The germination response to a range of KNO(3) concentrations was followed during preincubation at 24-hour intervals. During the entire 264-hour preincubation period increasingly higher nitrate concentrations were required to maintain a response. This resulted in a right-hand shift of the dose-response curve parallel to the x axis. After 120 hours the high maximum germination level started to decline. The dose-response curves could be simulated by an equation from the receptor-occupancy theory. It is proposed that induction of secondary dormancy is a result of a decrease of the number of nitrate receptors. After 24 and 48 hours of preincubation, the nitrate-response curves were biphasic. The biphasic character could be related to the level of endogenous nitrate and to a differential requirement for nitrate of two fractions of the seed population. Similarities with the behavior of fluence-response curves after prolonged dark incubation led to the hypothesis that phytochrome and nitrate share the same site of action.

Dual Effect of Light on the Gibberellin- and Nitrate-Stimulated Seed Germination of Sisymbrium officinale and Arabidopsis thaliana.

Red light (R) has a dual effect on the seed germination of the two related species Arabidopsis thaliana and Sisymbrium officinale. The two species provide different means to separate the light-effects. In S. officinale, stimulation of germination by R depends on the stimultaneous presence of nitrate (light-effect I). The effect of both factors is completely blocked by tetcyclacis, an inhibitor of gibberellin (GA)-biosynthesis. Addition of a mixture of gibberellins A(4) and A(7) (GA(4+7)) antagonizes the inhibition. In the absence of nitrate, R shifts germination to lower GA-requirement (light-effect II). In A. thaliana a similar second light-effect is seen on the GA-requirement of GA-deficient ga-1 mutant seeds. R stimulates germination of wild type seeds in water (light-effect I). For both species, light-effect I shows a fluence threshold value of approximately 10(-5) moles per square meter, which is independent of the nitrate concentration. Increasing nitrate concentrations narrow the fluence-range required for maximal germination whereby the product of nitrate concentration and fluence value determines the germination level, indicating a multiplicative interaction between R and nitrate. Fluence-response curves for light-effect II are similar for both species. Germination occurs in the range of 10(-6) to 10(-2) moles per square meter fluence. The maximal level of germination is determined by the level of dark-germination and light-effect II. Increasing GA(4+7) concentrations induce a shift to lower fluence values. It is shown that in the second effect the co-action of R and exogenous GA(4+7) is clearly additive. It is concluded that light-effect I induces a chain of events leading to GA biosynthesis. Light-effect II seems to enhance the sensitivity of the seeds to GAs.

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.

In Vivo Inhibition of Seed Development and Reserve Protein Accumulation in Recombinants of Abscisic Acid Biosynthesis and Responsiveness Mutants in Arabidopsis thaliana.

In Arabidopsis thaliana, seed development in recombinants of the ABA-deficient aba mutant with the ABA response mutants abi1 or abi3 is compared to wild type and the monogenic parents. Aberrant seed development occurred in the aba,abi3 recombinant and was normal in aba,abi1, abi3 and aba,abi1 seeds. Embryos of the recombinant aba,abi3 seeds maintained the green color until maturity, the seeds kept a high water content, did not form the late abundant 2S and 12S storage proteins, were desiccation intolerant, and often showed viviparous germination. Application of ABA, and particularly of an ABA analog, to the roots of plants during seed development partially alleviated the aberrant phenotype. Seeds of aba,abi3 were normal when they developed on a mother plant heterozygous for Aba. In contrast to seed development, the induction of dormancy was blocked in all monogenic mutants and recombinants. Dormancy was only induced by embryonic ABA; it could not be increased by maternal ABA or ABA applied to the mother plant. It is concluded that endogenous ABA has at least two different effects in developing seeds. The nature of these responses and of the ABA response system is discussed.

The second step of the biphasic endosperm cap weakening that mediates tomato (Lycopersicon esculentum) seed germination is under control of ABA.

The role of abscisic acid (ABA) in the weakening of the endosperm cap prior to radicle protrusion in tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds was studied. The endosperm cap weakened substantially in both water and ABA during the first 38 h of imbibition. After 38 h the force required for endosperm cap puncturing was arrested at 0.35 N in ABA, whereas in water a further decrease occurred until the radicle protruded. During the first 2 d of imbibition endo-beta-mannanase activity was correlated with the decrease in required puncture force and with the appearance of ice-crystal-induced porosity in the cell walls as observed by scanning electron microscopy. Prolonged incubation in ABA resulted in the loss of endo-beta-mannanase activity and the loss of ice-crystal-induced porosity, but not in a reversion of the required puncture force. ABA also had a distinct but minor effect on the growth potential of the embryo. However, endosperm cap resistance played the limiting role in the completion of germination. It was concluded that (a) endosperm cap weakening is a biphasic process and (b) inhibition of germination by ABA is through the second step in the endosperm cap weakening process.

Cell division and subsequent radicle protrusion in tomato seeds are inhibited by osmotic stress but DNA synthesis and formation of microtubular cytoskeleton are not.

We studied cell cycle events in embryos of tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds during imbibition in water and during osmoconditioning ("priming") using both quantitative and cytological analysis of DNA synthesis and beta-tubulin accumulation. Most embryonic nuclei of dry, untreated control seeds were arrested in the G(1) phase of the cell cycle. This indicated the absence of DNA synthesis (the S-phase), as confirmed by the absence of bromodeoxyuridine incorporation. In addition, beta-tubulin was not detected on western blots and microtubules were not present. During imbibition in water, DNA synthesis was activated in the radicle tip and then spread toward the cotyledons, resulting in an increase in the number of nuclei in G(2). Concomitantly, beta-tubulin accumulated and was assembled into microtubular cytoskeleton networks. Both of these cell cycle events preceded cell expansion and division and subsequent growth of the radicle through the seed coat. The activation of DNA synthesis and the formation of microtubular cytoskeleton networks were also observed throughout the embryo when seeds were osmoconditioned. However, this pre-activation of the cell cycle appeared to become arrested in the G(2) phase since no mitosis was observed. The pre-activation of cell cycle events in osmoconditioned seeds appeared to be correlated with enhanced germination performance during re-imbibition in water.

Endo-beta-mannanase activity increases in the skin and outer pericarp of tomato fruits during ripening.

Activity of endo-beta-mannanase increases during ripening of tomato (Lycopersicon esculentum Mill.) fruit of the cultivar Trust. beta-Mannoside mannohydrolase is also present during ripening, but its pattern of activity is different from that of endo-beta-mannanase. The increase in endo-beta-mannanase activity is greatest in the skin, and less in the outer and inner pericarp regions. This enzyme is probably bound to the walls of the outermost cell layers of the fruit during ripening, and it requires a high-salt buffer for effective extraction. The enzyme protein, as detected immunologically on Western blots, is present during the early stages of ripening, before any enzyme activity is detectable. The mRNA for the enzyme is also present at these stages; endo-beta-mannanase may be produced and sequestered in a mature-sized inactive form during early ripening. Most non-ripening mutants of tomato exhibit reduced softening and lower endo-beta-mannanase activity, but a cause-and-effect relationship between the enzyme and ripening is unlikely because some cultivars which ripen normally do not exhibit any endo-beta-mannanase activity in the fruit.