Flagellin perception varies quantitatively in Arabidopsis thaliana and its relatives

Much is known about the evolution of plant immunity components directed against specific pathogen strains: They show pervasive functional variation and have the potential to coevolve with pathogen populations. However, plants are effectively protected against most microbes by generalist immunity components that detect conserved pathogen-associated molecular patterns (PAMPs) and control the onset of PAMP-triggered immunity. In Arabidopsis thaliana, the receptor kinase flagellin sensing 2 (FLS2) confers recognition of bacterial flagellin (flg22) and activates a manifold defense response. To decipher the evolution of this system, we performed functional assays across a large set of A. thaliana genotypes and Brassicaceae relatives. We reveal extensive variation in flg22 perception, most of which results from changes in protein abundance. The observed variation correlates with both the severity of elicited defense responses and bacterial proliferation. We analyzed nucleotide variation segregating at FLS2 in A. thaliana and detected a pattern of variation suggestive of the rapid fixation of a novel adaptive allele. However, our study also shows that evolution at the receptor locus alone does not explain the evolution of flagellin perception; instead, components common to pathways downstream of PAMP perception likely contribute to the observed quantitative variation. Within and among close relatives, PAMP perception evolves quantitatively, which contrasts with the changes in recognition typically associated with the evolution of R genes.     

Evolution of interspecies unilateral incompatibility in the relatives of Arabidopsis thaliana

The evolutionary concurrence of intraspecies self‐incompatibility (SI) and explosive angiosperm radiation in the Cretaceous have led to the hypothesis that SI was one of the predominant drivers of rapid speciation in angiosperms. Interspecies unilateral incompatibility (UI) usually occurs when pollen from a self‐compatible (SC) species is rejected by the pistils of a SI species, while the reciprocal pollination is compatible (UC). Although this SI × SC type UI is most prevalent and viewed as a prezygotic isolation barrier to promote incipient speciation of angiosperms, comparative evidence to support such a role is lacking. We show that SI × SI type UI in SI species pairs is also common in the well‐characterized accessions representing the four major lineages of the Arabidopsis genus and is developmentally regulated. This allowed us to reveal a strong correlation between UI strength and species divergence in these representative accessions. In addition, analyses of a SC accession and the pseudo‐self‐compatible (psc) spontaneous mutant of Arabidopsis lyrata indicate that UI shares, at least, common pollen rejection pathway with SI. Furthermore, genetic and genomic analyses of SI × SI type UI in A. lyrata × A. arenosa species pair showed that two major‐effect quantitative trait loci are the stigma and pollen‐side determinant of UI, respectively, which could be involved in heterospecies pollen discrimination. By revealing a close link between UI and SI pathway, particularly between UI and species divergence in these representative accessions, our findings establish a connection between SI and speciation. Thus, the pre‐existence of SI system would have facilitated the evolution of UI and accordingly promote speciation.     

Signals of speciation within Arabidopsis thaliana in comparison with its relatives

The species within the now well-defined Arabidopsis genus provide biological materials suitable to investigate speciation and the development of reproductive isolation barriers between related species. Even within the model species A. thaliana, genetic differentiation between populations due to environmental adaptation or demographic history can lead to cases where hybrids between accessions are non-viable. Experimental evidence supports the importance of genome duplications and genetic epistatic interactions in the occurrence of reproductive isolation. Other examples of adaptation to specific environments can be found in Arabidopsis relatives where hybridization and chromosome doubling lead to new amphidiploid species. Molecular signals of speciation found in the Arabidopsis genus should provide a better understanding of speciation processes in plants from a genetic, molecular and evolutionary perspective.     

Lesser known relatives of miRNA

Since the discovery of RNAi (RNA interference) major attention has focused on studying miRNA (microRNA) and siRNA (small interfering RNA). However, within the last few years, several other small ncRNAs (non-coding RNAs) have been discovered and thus various newer acronyms representing these ‘other’ classes of small ncRNAs have populated the literature. Of these, piRNA (Piwi-interacting RNA) has been gaining importance because of its role as the guardian of the germline genome. Some of the other newly discovered small ncRNAs have been mostly reported in plants, and they are yet to be studied more comprehensively. Nevertheless, piRNA and the ‘other’ small ncRNAs deserve some discussion because they are members of the increasingly large family of small ncRNAs.     

Comparing the linkage maps of the close relatives Arabidopsis lyrata and A. thaliana

We have constructed a genetic map of Arabidopsis lyrata, a self-incompatible relative of the plant model species A. thaliana. A. lyrata is a diploid (n = 8) species that diverged from A. thaliana (n = 5) approximately 5 MYA. Mapping was conducted in a full-sib progeny of two unrelated F(1) hybrids between two European populations of A. lyrata ssp. petraea. We used the least-squares method of the Joinmap program for map construction. The gross chromosomal differences between the two species were most parsimoniously explained with three fusions, two reciprocal translocations, and one inversion. The total map length was 515 cM, and the distances were 12% larger than those between corresponding markers in the linkage map of A. thaliana. The 72 markers, consisting of microsatellites and gene-based markers, were spaced on average every 8 cM. Transmission ratio distortion was extensive, and most distortions were specific to each reciprocal cross, suggesting cytoplasmic interactions. We estimate locations and most probable genotype frequencies of transmission ratio distorting loci (TRDL) with a Bayesian method and discuss the possible reasons for the observed distortions.     

Evolution of microsatellites in Arabis petraea and Arabis lyrata, outcrossing relatives of Arabidopsis thaliana

We examined microsatellite variation in two diploid, outcrossing relatives of Arabidopsis thaliana, Arabis petraea and Arabis lyrata. The primer sequences were derived from A. thaliana. About 50% (14 loci) of the A. thaliana primers could successfully amplify microsatellites in the related species. Analysis of microsatellite structure in the related species showed that there had been large changes in the microsatellites: there were large differences in repeat numbers and many of the A. thaliana simple repeats were shorter in the related species. For the loci we compared, the related species had a much lower level of variability at the microsatellites than Japanese wild populations of A. thaliana. This is presumably related to the different microsatellite structures, because allozyme data showed that the outcrossing relatives were highly polymorphic compared to other outcrossing herbaceous species. Use of microsatellites in assessing variability or phylogenetic relationships between different species requires caution, because changes in microsatellite structure may alter evolutionary rates.      

Geographic and evolutionary diversification of glucosinolates among near relatives of Arabidopsis thaliana (Brassicaceae)

Glucosinolates are biologically active secondary metabolites that display both intra- and interspecific variation in the order Brassicales. Glucosinolate profiles have not been interpreted within a phylogenic framework and little is known regarding the processes that influence the evolution of glucosinolate diversity at a macroevolutionary scale. We have analyzed leaf glucosinolate profiles from members of the Brassicaceae that have diverged from Arabidopsis thaliana within the last 15 million years and interpreted our findings relative to the phylogeny of this group. We identified several interspecific polymorphisms in glucosinolate composition. A majority of these polymorphisms are lineage-specific secondary losses of glucosinolate characters, but a gain-of-character polymorphism was also detected. The genetic basis of most observed polymorphisms appears to be regulatory. In the case of A. lyrata, geographic distribution is also shown to contribute to glucosinolate metabolic diversity. Further, we observed evidence of gene-flow between sympatric species, parallel evolution, and the existence of genetic constraints on the evolution of glucosinolates within the Brassicaceae.     

Conservation and clade-specific diversification of pathogen-inducible tryptophan and indole glucosinolate metabolism in Arabidopsis thaliana relatives

? A hallmark of the innate immune system of plants is the biosynthesis of low-molecular-weight compounds referred to as secondary metabolites. Tryptophan-derived branch pathways contribute to the capacity for chemical defense against microbes in Arabidopsis thaliana. ? Here, we investigated phylogenetic patterns of this metabolic pathway in relatives of A. thaliana following inoculation with filamentous fungal pathogens that employ contrasting infection strategies. ? The study revealed unexpected phylogenetic conservation of the pathogen-induced indole glucosinolate (IG) metabolic pathway, including a metabolic shift of IG biosynthesis to 4-methoxyindol-3-ylmethylglucosinolate and IG metabolization. By contrast, indole-3-carboxylic acid and camalexin biosyntheses are clade-specific innovations within this metabolic framework. A Capsella rubella accession was found to be devoid of any IG metabolites and to lack orthologs of two A. thaliana genes needed for 4-methoxyindol-3-ylmethylglucosinolate biosynthesis or hydrolysis. However, C. rubella was found to retain the capacity to deposit callose after treatment with the bacterial flagellin-derived epitope flg22 and pre-invasive resistance against a nonadapted powdery mildew fungus. ? We conclude that pathogen-inducible IG metabolism in the Brassicaceae is evolutionarily ancient, while other tryptophan-derived branch pathways represent relatively recent manifestations of a plant-pathogen arms race. Moreover, at least one Brassicaceae lineage appears to have evolved IG-independent defense signaling and/or output pathway(s).     

Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of known cyclophilin proteins

We have isolated four members of the Arabidopsis cyclophilin (CyP) gene family, designated ROC1 to ROC4 (rotamase CyP). Deduced peptides of ROC1, 2 and 3 are 75% to 91% identical to Brassica napus cytosolic CyP, contain no leader peptides and include a conserved seven amino-acid insertion relative to mammalian cytosolic CyPs. Two other Arabidopsis CyPs, ROC5 (43H1; ATCYP1) and ROC6 (ATCYP2), share these features. ROC1, ROC2, ROC3 and ROC5 are expressed in all tested organs of light-grown plants. ROC2 and ROC5 show elevated expression in flowers. Expression of ROC1, ROC2, and ROC3 decreases in darkness and these genes also exhibit small elevations in expression upon wounding. The five Arabidopsis genes encoding putative cytosolic CyPs (ROC1, 2, 3, 5 and 6) contain no introns. In contrast, ROC4, which encodes a chloroplast stromal CyP, is interrupted by six introns. ROC4 is not expressed in roots, and is strongly induced by light. Phylogenetic trees of all known CyPs and CyP-related proteins provide evidence of possible horizontal transfer of CyP genes between prokaryotes and eukaryotes and of a possible polyphyletic origin of these proteins within eukaryotes. These trees also show significant grouping of eukaryotic CyPs on the basis of subcellular localization and structure. Mitochondrial CyPs are closely related to cytosolic CyPs of the source organism, but endoplasmic reticulum CyPs form separate clades. Known plant CyPs fall into three clades, one including the majority of higher-plant cytosolic CyPs, one including only ROC2 and a related rice CyP, and one including only chloroplast CyPs.     

Circumnutations of Arabidopsis thaliana Seedlings

The hypocotyls of Arabidopsis thaliana seedlings exhibit circumnutations with different frequency ranges. Using a picture-analysis system, two types of oscillations were distinguished, short period (SPN) and long period nutations (LPN). The period of the SPNs is between 20 and 60 min, decreases with increasing temperature (between 20° and 30°C; Q 10 of 2.0) and increases with age. The SPNs changed frequently between circular and pendular movements. The circumnutation usually occurred clockwise, but the direction could change. For SPNs to occur, a minimum growth rate of 0.05 mm/h was required. The period of the LPNs ranged from 1 to 8 h. Peaks occurred around 95 and 200 min. The movements of the LPNs are mostly counterclockwise. The oscillation could change from circular to elliptic or pendular. In contrast to SPNs, LPNs were found also at low growth rates. Under red light and in the strain Landsberg erecta the circumnutations were especially slow. Short and long period oscillations can occur simultaneously or separately in different plants. In cases of simultaneous occurrence the quotient between the period lengths of the LPN and SPN was not constant. The number of occurrences of these frequencies depended on the strain and on external factors such as temperature and light. In continuous bright white light phases of circumnutations alternated with quiescent periods. This behaviour was circadian and correlated with growth bursts.     

Circumnutations of Arabidopsis thaliana Seedlings

The hypocotyls of Arabidopsis thaliana seedlings exhibit circumnutations with different frequency ranges. Using a picture-analysis system, two types of oscillations were distinguished, short period (SPN) and long period nutations (LPN). The period of the SPNs is between 20 and 60 min, decreases with increasing temperature (between 20° and 30°C; Q 10 of 2.0) and increases with age. The SPNs changed frequently between circular and pendular movements. The circumnutation usually occurred clockwise, but the direction could change. For SPNs to occur, a minimum growth rate of 0.05 mm/h was required. The period of the LPNs ranged from 1 to 8 h. Peaks occurred around 95 and 200 min. The movements of the LPNs are mostly counterclockwise. The oscillation could change from circular to elliptic or pendular. In contrast to SPNs, LPNs were found also at low growth rates. Under red light and in the strain Landsberg erecta the circumnutations were especially slow. Short and long period oscillations can occur simultaneously or separately in different plants. In cases of simultaneous occurrence the quotient between the period lengths of the LPN and SPN was not constant. The number of occurrences of these frequencies depended on the strain and on external factors such as temperature and light. In continuous bright white light phases of circumnutations alternated with quiescent periods. This behaviour was circadian and correlated with growth bursts.     

Embryonic mutants of Arabidopsis thaliana

Genetic analysis of plant em-bryogenesis has been approached in part through the isolation and characterization of recessive embryonic mutants. The most extensive studies have dealt with maize and Arabidopsis. The high frequency of mutants defective in plant embryogenesis is consistent with the presence of many target genes with essential functions at this stage of the life cycle. Some mutants are likely to be defective in genes with general housekeeping functions. Others should facilitate the identification of genes with a more direct role in the regulation of morphogesis. Over 300 embryonic mutants of Arabidopsis isolated following chemical mutagenesis and T-DNA insertional mutagenesis are currently being analyzed. This collection includes embryonic le-thals, defectives, and pattern mutants. Developmental abnormalities include the presence of fused cotyledons, twin embryos, abnormally large suspensors, distorted epidermal layers, single cotyledons, enlarged shoot apices, pattern deletions and duplications, embryos with altered patterns of symmetry, bloated embryos with giant vacuolated cells, reduced hypocotyls that fail to produce roots, and embryos that protrude through the seed coat late in maturation. This review describes the isolation and characterization of embryonic mutants of Arabidopsis and their potential application to plant biology. © 1992 Wiley-Liss, Inc.     

Cardiolipin synthase of Arabidopsis thaliana

Functional expression studies in microorganisms showed that the Arabidopsis thaliana gene At4g04870 represents the cardiolipin synthase (CLS) gene encoding a hydrophobic preprotein of 38 kDa with a cleavable signal peptide for the import into mitochondria. CLS of Arabidopsis over-expressed in Escherichia coli has an alkaline pH optimum, a strict requirement for divalent cations and a distinctly lower K(m) for cytidinediphosphate-diacylglycerol than for phosphatidylglycerol. It displayed a preference for both its substrates esterified with unsaturated acyl groups. Solubilization and purification experiments revealed that the protein requires a defined phospholipid environment, particularly the presence of cardiolipin, to acquire its catalytically active conformation.     

Cadmium-Sensitive Mutants of Arabidopsis thaliana

A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (γ-glutamylcysteine)_n-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd.