Measuring spatial variation in natural selection using randomly-sown seeds of Arabidopsis thaliana

We describe a simple method of measuring spatial variation in fitness using randomly sown seeds. Given a random initial distribution of seeds, any spatial variation in the final distribution of genotypes must be the result of spatial variation in natural selection. Departures from spatial randomness are tested using a modification of join-count statistics, based on the probability that two randomly chosen plants separated by a given distance carry the same genetic marker. Monte Carlo simulations showed that for the randomly sown seeds design the join-count test had higher power to detect spatial variation in fitness than analysis of variance. The power was relatively independent of the number of genotypes used or the number of microsites sampled. The method was tested in the field using three standard lines of Arabidopsis thaliana. At the end of the life cycle, adjacent plants were significantly more likely to be the same line than randomly chosen pairs and the spatial dependence extended to distances of nearly 50 cm. Simple experiments like this may be especially useful for “comparative ecological genetics”, allowing systematic studies of natural selection among many species or habitats.     

Structure of flower in Arabidopsis thaliana: spatial pattern formation

Morphological analysis of flowers was carried out in Arabidopsis thaliana wild type plants and agamous and apetala2 mutants. No direct substitution of organs takes place in the mutants, since the number and position of organs in them do not correspond to the structure of wild type flower. In order to explain these data, a notion of spatial pattern formation in the meristem was introduced, which preceded the processes of appearance of organ primordia and formation of organs. Zones of acropetal and basipetal spatial pattern formation in the flower of wild type plants were postulated. It was shown that the acropetal spatial pattern formation alone took place in agamous mutants and basipetal spatial pattern formation alone, in apetala2 mutants. Different variants of flower structure are interpreted as a result of changes in the volume of meristem (space) and order of spatial pattern formation (time).     

Spatio-temporal heterogeneity in Arabidopsis thaliana leaves under drought stress

Using chlorophyll (chl) fluorescence imaging, we studied the effect of mild (MiDS), moderate (MoDS) and severe (SDS) drought stress on photosystem II (PSII) photochemistry of 4-week-old Arabidopsis thaliana. Spatio-temporal heterogeneity in all chl fluorescence parameters was maintained throughout water stress. After exposure to drought stress, maximum quantum yield of PSII photochemistry (F(v)/F(m)) and quantum efficiency of PSII photochemistry (Φ(PSΙΙ)) decreased less in the proximal (base) than in the distal (tip) leaf. The chl fluorescence parameter F(v) /F(m) decreased less after MoDS than MiDS. Under MoDS, the antioxidant mechanism of A. thaliana leaves seemed to be sufficient in scavenging reactive oxygen species, as evident by the decreased lipid peroxidation, the more excitation energy dissipated by non-photochemical quenching (NPQ) and decreased excitation pressure (1-q(p)). Arabidopsis leaves appear to function normally under MoDS, but do not seem to have particular metabolic tolerance mechanisms under MiDS and SDS, as revealed by the level of lipid peroxidation and decreased quantum yield for dissipation after down-regulation in PSII (Φ(NPQ)), indicating that energy dissipation by down-regulation did not function and electron transport (ETR) was depressed. The simultaneous increased quantum yield of non-regulated energy dissipation (Φ(NO)) indicated that both the photochemical energy conversion and protective regulatory mechanism were insufficient. The non-uniform photosynthetic pattern under drought stress may reflect different zones of leaf anatomy and mesophyll development. The data demonstrate that the effect of different degrees of drought stress on A. thaliana leaves show spatio-temporal heterogeneity, implying that common single time point or single point leaf analyses are inadequate.     

Physiological and molecular mechanisms underlying the integration of light and temperature cues in Arabidopsis thaliana seeds

The relief of dormancy and the promotion of seed germination are of extreme importance for a successful seedling establishment. Although alternating temperatures and light are signals promoting the relief of seed dormancy, the underlying mechanisms of their interaction in seeds are scarcely known. By exposing imbibed Arabidopsis thaliana dormant seeds to two‐day temperature cycles previous of a red light pulse, we demonstrate that the germination mediated by phytochrome B requires the presence of functional PSEUDO‐RESPONSE REGULATOR 7 (PRR7) and TIMING OF CAB EXPRESSION 1 (TOC1) alleles. In addition, daily cycles of alternating temperatures in darkness reduce the protein levels of DELAY OF GERMINATION 1 (DOG1), allowing the expression of TOC1 to induce seed germination. Our results suggest a functional role for some components of the circadian clock related with the action of DOG1 for the integration of alternating temperatures and light signals in the relief of seed dormancy. The synchronization of germination by the synergic action of light and temperature through the activity of circadian clock might have ecological and adaptive consequences.     

Supercooling capacity of seeds and seedlings in Arabidopsis thaliana.

The influence of the water content of seeds and seedlings of Arabidopsis thaliana (Ecotype Columbia:2) on their supercooling capacity was investigated. Equilibration of the seeds to various air relative humidities resulted in final moisture contents ranging from 8 to 82% (dry weight basis). No supercooling point could be detected when the water content remained below 32.5%, and in seeds at just above this moisture level ice crystals started to form at -26 degrees C. However, cooling partly affected the germination of seeds down to a water content of 26.5%. Upon imbibition, the supercooling point of the seeds remained around -21.6 degrees C and rose sharply to -14.7 degrees C when visible germination started. It remained around -13 degrees C during the following 96 h while the water content of the seedlings increased from 155 to 870%. Hydrated seeds (above 32.5% water content), germinated seeds, and seedlings of Arabidopsis cannot survive being frozen.     

Novel whole-mount FISH analysis for intact root of Arabidopsis thaliana with spatial reference to 3D visualization

Journal of Plant Research - Whole-mount fluorescent in situ hybridization (WM-FISH) is an effective tool to observe chromosome behavior in tissues or organs. However, it is difficult to obtain a...     

A simple and rapid method for visualization of male meiotic chromosomes in Arabidopsis thaliana

Meiotic chromosomes are of basic interest to the geneticist and cell biologist who study their behavior. A rapid and highly repeatable method for visualization of meiotic chromosomes is useful. Here we describe a fast staining protocol for Arabidopsis male meiotic chromosomes. Meiocytes were squashed into a labeling buffer, the chromosome morphology could be analyzed using fluorescence without any additional treatment.     

Production of hydroxy fatty acids in the seeds of Arabidopsis thaliana

Seed-specific expression in Arabidopsis thaliana of oleate hydroxylase enzymes from castor bean and Lesquerella fendleri resulted in the accumulation of hydroxy fatty acids in the seed oil. By using various Arabidopsis mutant lines it was shown that the endoplasmic reticulum (ER) n-3 desaturase (FAD3) and the FAE1 condensing enzyme are involved in the synthesis of polyunsaturated and very-long-chain hydroxy fatty acids, respectively. In Arabidopsis plants with an active ER Delta12-oleate desaturase the presence of hydroxy fatty acids corresponded to an increase in the levels of 18:1 and a decrease in 18:2 levels. Expression in yeast indicates that the castor hydroxylase also has a low level of desaturase activity.     

A fast brassinolide-regulated response pathway in the plasma membrane of Arabidopsis thaliana

To understand molecular processes in living plant cells, quantitative spectro-microscopic technologies are required. By combining fluorescence lifetime spectroscopy with confocal microscopy, we studied the subcellular properties and function of a GFP-tagged variant of the plasma membrane-bound brassinosteroid receptor BRI1 (BRI1-GFP) in living cells of Arabidopsis seedlings. Shortly after adding brassinolide, we observed BRI1-dependent cell-wall expansion, preceding cell elongation. In parallel, the fluorescence lifetime of BRI1-GFP decreased, indicating an alteration in the receptor's physico-chemical environment. The parameter modulating the fluorescence lifetime of BRI1-GFP was found to be BL-induced hyperpolarization of the plasma membrane. Furthermore, for induction of hyperpolarization and cell-wall expansion, activation of the plasma membrane P-ATPase was necessary. This activation required BRI1 kinase activity, and was mediated by BL-modulated interaction of BRI1 with the P-ATPase. Our results were used to develop a model suggesting that there is a fast BL-regulated signal response pathway within the plasma membrane that links BRI1 with P-ATPase for the regulation of cell-wall expansion.     

Effects of gene expression on molecular evolution in Arabidopsis thaliana and Arabidopsis lyrata

We analyzed the complete genome sequence of Arabidopsis thaliana and sequence data from 83 genes in the outcrossing A. lyrata, to better understand the role of gene expression on the strength of natural selection on synonymous and replacement sites in Arabidopsis. From data on tRNA gene abundance, we find a good concordance between codon preferences and the relative abundance of isoaccepting tRNAs in the complete A. thaliana genome, consistent with models of translational selection. Both EST-based and new quantitative measures of gene expression (MPSS) suggest that codon preferences derived from information on tRNA abundance are more strongly associated with gene expression than those obtained from multivariate analysis, which provides further support for the hypothesis that codon bias in Arabidopsis is under selection mediated by tRNA abundance. Consistent with previous results, analysis of protein evolution reveals a significant correlation between gene expression level and amino acid substitution rate. Analysis by MPSS estimates of gene expression suggests that this effect is primarily the result of a correlation between the number of tissues in which a gene is expressed and the rate of amino acid substitution, which indicates that the degree of tissue specialization may be an important determinant of the rate of protein evolution in Arabidopsis.     

Phosphatidic acid is a major phospholipid class in reproductive organs of Arabidopsis thaliana

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.     

Biphasic fluence-response curves for light induced germination of Arabidopsis thaliana seeds

Abstract With appropriate pretreatment of the seeds fluence-response curves for the induction of germination of Arabidopsis thaliana show two phases. A proportion of the population responds to very low fluence (VLFR), 10⁴–10^?2μmolm^?2 establishing 10^?4–10^?2% of the total phytochrome in the far-red absorbing form (Pfr) and a proportion of the population respond to low fluence (LFR), 1–1000 μmolm^?2, establishing 1–75% Pfr. The VLFR is nol normally seen because the pre-existing Pfr level satisfies the Pfr requirement or use of green safelight establishes more Pfr than necessary to saturate the VLFR. Endogenous Pfr was depicted by a 24 h 35°C treatment, presumably as a result of dark destruction and/or dark reversion to the red absorbing form of phytochrome (Pr), making it possible to visualize the VLFR. A short pulse of 35°C treatment in combination with an appropriate temperature regime is also able to sensitize a proportion of the seed population. The proportion of the population showing the VLFR is determined by the duration of the cold imbibition pretreatment as well as the duration of the 35°C treatment. Complex fluence-response curves were observed in which a proportion of the seeds being promoted in the VLFR range, were inhibited at higher fluences before being further promoted in the LFR range. This was particularly clear for seed batches being sensitized by a short 35°C treatment. The VLFR may be of significance in the natural environment, enabling seeds buried in the upper layer of the soil to germinate, where the fluence rate falls off sharply and the LFR is not satisfied. A model is presented to explain the two phases in the fluence-response curves.     

The lipid polyester composition of Arabidopsis thaliana and Brassica napus seeds

Mature seeds of Arabidopsis thaliana and Brassica napus contain a complex mixture of aliphatic monomers derived from the non-extractable lipid polyesters deposited by various seed tissues. Methods of polyester depolymerization of solvent-extracted seeds and analysis of aliphatic monomers were compared. Sodium methoxide-catalyzed depolymerization, followed by GC analysis of the acetylated monomers, was developed for routine quantitative analysis suitable for 0.5g seed samples. In Arabidopsis seeds, the major C16 and C18 monomers identified included omega-hydroxy fatty acids and alpha,omega-dicarboxylic acids derived from palmitate, oleate and linoleate, and 9,10,18-trihydroxyoctadecenoic acid. Among monomers which can collectively be considered likely to be derived from suberin, docosan-1-ol, docosane-1,22-diol, 22-hydroxydocosanoic acid, 24-hydroxytetracosanoic acid, tetracosane-1,24-dioic acid and ferulic acid were the major species. Compared to Arabidopsis, Brassica seeds showed a roughly similar proportion of monomer classes, with the exception that alkan-1ols were 3-fold higher. Also, there were much less C24 aliphatic species and significant amounts of C14-C16 alkan-1ols, including iso- and anteiso-methyl branched compounds. Dissection and analysis of mature Brassica seeds showed that the trihydroxy C18:1 fatty acid was found mainly in the embryo, while ferulate, fatty alcohols and C22 and C24 species were specific to the seed coat plus endosperm.