Cross-talk between singlet oxygen- and hydrogen peroxide-dependent signaling of stress responses in Arabidopsis thaliana

Upon a dark-to-light shift, the conditional fluorescent (flu) mutant of Arabidopsis releases singlet oxygen (1O2) within the plastid compartment. Distinct sets of nuclear genes are activated that are different from those induced by superoxide (O2*-)) and/or hydrogen peroxide (H2O2), suggesting that different types of reactive oxygen species activate distinct signaling pathways. It is not known whether the pathways operate separately or interact with each other. We have addressed this problem by modulating noninvasively the level of H2O2 in plastids by means of a transgenic line that overexpresses the thylakoid-bound ascorbate peroxidase (tAPX). The overexpression of the H2O2-specific scavenger reduced strongly the activation of nuclear genes in plants treated with the herbicide paraquat that in the light leads to the enhanced generation of O2*- and H2O2. In the flu mutant overexpressing tAPX, the intensity of 1O2-mediated cell death and growth inhibition was increased when compared with the flu parental line. Also, the expression of most of the nuclear genes that were rapidly activated after the release of 1O2 was significantly higher in flu plants overexpressing tAPX, whereas in wild-type plants, overexpression of tAPX did not lead to visible stress responses and had only a very minor impact on nuclear gene expression. The results suggest that H2O2 antagonizes the 1O2-mediated signaling of stress responses as seen in the flu mutant. This cross-talk between H2O2- and 1O2-dependent signaling pathways might contribute to the overall stability and robustness of wild-type plants exposed to adverse environmental stress conditions.     

Genetics of microenvironmental canalization in Arabidopsis thaliana

Canalization is a fundamental feature of many developmental systems, yet the genetic basis for this property remains elusive. We examine the genetic basis of microenvironmental canalization in the model plant Arabidopsis thaliana, focusing on differential developmental stability between genotypes in one fitness and four quantitative morphological traits. We measured developmental stability in genetically identical replicates of two populations of recombinant inbred (RI) lines and one population of geographically widespread accessions of A. thaliana grown in two different photoperiod-controlled environments. We were able to map quantitative trait loci associated with developmental stability. We also identified a candidate gene, ERECTA, that may contribute to microenvironmental canalization in rosette leaf number under long-day photoperiods, and analysis of mutant lines indicates that the er-105 allele results in increased canalization for this trait. ERECTA, which encodes a signaling protein, appears to act as an ecological amplifier by transducing developmental noise (e.g., microenvironmental variation) into phenotypic differentiation. We also measured genotypic selection on four plant architecture traits and find evidence for selection for both increased and decreased canalization at various traits.     

A unique mechanism for protein processing and degradation in Arabidopsis thaliana

Precursor protease vesicles are plant-specific compartments containing precursors of enzymes that are thought to participate in the degradation of cellular components in organs undergoing senescence. We report in vivo evidence that the precursor protease vesicle-localized vacuolar processing enzyme-gamma (VPEgamma) is critical for maturation of the plant vacuolar protease AtCPY. We also provide biochemical and functional evidence that VPEgamma is involved in degradation of the vacuolar invertase AtFruct4 in aging tissues. Moreover, a proteomics-based approach identified various proteins found in the vacuoles of aging vpegamma mutants but not in WT plants, suggesting a unique role of VPEgamma in protein processing and degradation in Arabidopsis.     

Male-biased transmission of deleterious mutations to the progeny in Arabidopsis thaliana

The extent and cause of male-biased mutation rates, the higher number of mutations in sperm than in eggs, is currently an active and controversial subject. Recent evidence indicates that this male (sperm) bias not only occurs in animals but also in plants. The higher mutation rate in plant sperm was inferred from rates of evolution of neutral DNA regions, and the results were confined to the mitochondria and chloroplasts of gymnosperms. However, the relative transmission rates of deleterious mutations, which have substantial evolutionary consequences, have rarely been studied. Here, an investigation is described by using the hermaphroditic self-compatible flowering plant Arabidopsis thaliana, in which we artificially increased the rate of mutation in pollen (i.e., sperm donor) and maternal (i.e., egg donor) parents, by using two kinds of UV irradiation in parallel and separate experiments, and assessed the deleterious effects on fitness of the F(2) generation. The results show that more deleterious induced mutations are transmitted to the progeny by a sperm than by an egg. These findings provide the first experimental evidence that more deleterious mutations are inherited from sperm than from an egg in any organism. Possible causes underlying this male bias are discussed.     

Vacuolar sorting receptor for seed storage proteins in Arabidopsis thaliana

The seeds of higher plants accumulate large quantities of storage protein. During seed maturation, storage protein precursors synthesized on rough endoplasmic reticulum are sorted to protein storage vacuoles, where they are converted into the mature forms and accumulated. Previous attempts to determine the sorting machinery for storage proteins have not been successful. Here we show that a type I membrane protein, AtVSR1/AtELP, of Arabidopsis functions as a sorting receptor for storage proteins. The atvsr1 mutant missorts storage proteins by secreting them from cells, resulting in an enlarged and electron-dense extracellular space in the seeds. The atvsr1 seeds have distorted cells and smaller protein storage vacuoles than do WT seeds, and atvsr1 seeds abnormally accumulate the precursors of two major storage proteins, 12S globulin and 2S albumin, together with the mature forms of these proteins. AtVSR1 was found to bind to the C-terminal peptide of 12S globulin in a Ca2+-dependent manner. These findings demonstrate a receptor-mediated transport of seed storage proteins to protein storage vacuoles in higher plants.     

Transposable elements and small RNAs contribute to gene expression divergence between Arabidopsis thaliana and Arabidopsis lyrata

Transposable elements (TEs) are often the primary determinant of genome size differences among eukaryotes. In plants, the proliferation of TEs is countered through epigenetic silencing mechanisms that prevent mobility. Recent studies using the model plant Arabidopsis thaliana have revealed that methylated TE insertions are often associated with reduced expression of nearby genes, and these insertions may be subject to purifying selection due to this effect. Less is known about the genome-wide patterns of epigenetic silencing of TEs in other plant species. Here, we compare the 24-nt siRNA complement from A. thaliana and a closely related congener with a two- to threefold higher TE copy number, Arabidopsis lyrata. We show that TEs--particularly siRNA-targeted TEs--are associated with reduced gene expression within both species and also with gene expression differences between orthologs. In addition, A. lyrata TEs are targeted by a lower fraction of uniquely matching siRNAs, which are associated with more effective silencing of TE expression. Our results suggest that the efficacy of RNA-directed DNA methylation silencing is lower in A. lyrata, a finding that may shed light on the causes of differential TE proliferation among species.     

Regulation of floral organ abscission in Arabidopsis thaliana

Abscission is a developmental program that results in the active shedding of infected or nonfunctional organs from a plant body. Here, we establish a signaling pathway that controls abscission in Arabidopsis thaliana from ligand, to receptors, to downstream effectors. Loss of function mutations in Inflorescence Deficient in Abscission (IDA), which encodes a predicted secreted small protein, the receptor-like protein kinases HAESA (HAE) and HAESA-like 2 (HSL2), the Mitogen-Activated Protein Kinase Kinase 4 (MKK4) and MKK5, and a dominant-negative form of Mitogen-Activated Protein Kinase 6 (MPK6) in a mpk3 mutant background all have abscission-defective phenotypes. Conversely, expression of constitutively active MKKs rescues the abscission-defective phenotype of hae hsl2 and ida plants. Additionally, in hae hsl2 and ida plants, MAP kinase activity is reduced in the receptacle, the part of the stem that holds the floral organs. Plants overexpressing IDA in a hae hsl2 background have abscission defects, indicating HAE and HSL2 are epistatic to IDA. Taken together, these results suggest that the sequential action of IDA, HAE and HSL2, and a MAP kinase cascade regulates the programmed separation of cells in the abscission zone.     

Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana

We used hybridization to the ATH1 gene expression array to interrogate genomic DNA diversity in 23 wild strains (accessions) of Arabidopsis thaliana (arabidopsis), in comparison with the reference strain Columbia (Col). At <1% false discovery rate, we detected 77,420 single-feature polymorphisms (SFPs) with distinct patterns of variation across the genome. Total and pair-wise diversity was higher near the centromeres and the heterochromatic knob region, but overall diversity was positively correlated with recombination rate (R(2) = 3.1%). The difference between total and pair-wise SFP diversity is a relative measure contrasting diversifying or frequency-dependent selection, similar to Tajima's D, and can be calibrated by the empirical genome-wide distribution. Each unique locus, centered on a gene, has a diversity and selection score that suggest a relative role in past evolutionary processes. Homologs of disease resistance (R) genes include members with especially high levels of diversity often showing frequency-dependent selection and occasionally evidence of a past selective sweep. Receptor-like and S-locus proteins also contained members with elevated levels of diversity and signatures of selection, whereas other gene families, bHLH, F-box, and RING finger proteins, showed more typical levels of diversity. SFPs identified with the gene expression array also provide an empirical hybridization polymorphism background for studies of gene expression polymorphism and are available through the genome browser http://signal.salk.edu/cgi-bin/AtSFP.     

PIEZO ion channel is required for root mechanotransduction in Arabidopsis thaliana

Plant roots adapt to the mechanical constraints of the soil to grow and absorb water and nutrients. As in animal species, mechanosensitive ion channels in plants are proposed to transduce external mechanical forces into biological signals. However, the identity of these plant root ion channels remains unknown. Here, we show that Arabidopsis thaliana PIEZO1 (PZO1) has preserved the function of its animal relatives and acts as an ion channel. We present evidence that plant PIEZO1 is expressed in the columella and lateral root cap cells of the root tip, which are known to experience robust mechanical strain during root growth. Deleting PZO1 from the whole plant significantly reduced the ability of its roots to penetrate denser barriers compared to wild-type plants. pzo1 mutant root tips exhibited diminished calcium transients in response to mechanical stimulation, supporting a role of PZO1 in root mechanotransduction. Finally, a chimeric PZO1 channel that includes the C-terminal half of PZO1 containing the putative pore region was functional and mechanosensitive when expressed in naive mammalian cells. Collectively, our data suggest that Arabidopsis PIEZO1 plays an important role in root mechanotransduction and establish PIEZOs as physiologically relevant mechanosensitive ion channels across animal and plant kingdoms.

Plants extend roots within the soil to access water and nutrients as well as provide stability for the aerial parts of the plant. Underground barriers caused by drought and/or heterogeneous soil components can exert mechanical resistance that alters root extension and penetration (1–3). The root cap at the very tip of the primary root is a dynamic organ that contains different classes of stem cells that divide asymmetrically and is essential for growth through harder media and soils (4). Bending or poking root tips elicits a transient Ca²⁺ influx with short latency that is blocked by lanthanides, including Gd³⁺, a nonselective inhibitor of mechanically activated (MA) cation channels (5–7). However, the molecular identity of putative ion channels underlying this response is unknown. Only a few mechanosensitive ion channels have been described in plants (8). MSL8 plays a mechanosensory role in pollen (9), MSL10 is involved in cell swelling (8, 10), OSCA1 has mainly been characterized for its role in osmosensation (11), and OSCA1.3 regulates stomatal closure during immune signaling (12). It has been proposed that MCA1, expressed in the elongation zone but not the root cap, is a stretch-activated calcium permeable ion channel involved in soil penetration; however, evidence for its being a bona fide ion channel capable of detecting mechanical force is lacking (13–15). Recently, it has been shown that mechanosensitive Ca²⁺ channel activity is dependent on the developmental regulator DEK1; however, whether DEK1 is a pore-forming ion channel has not yet been addressed (16, 17). The genome of Arabidopsis thaliana encodes an ortholog of the mammalian mechanosensitive ion channels PIEZO1 and PIEZO2 (18). Given that PIEZOs play prominent roles in multiple aspects of animal mechanosensation and physiology (19–22), we investigated the role of A. thaliana PIEZO1 (PZO1) in plant mechanosensation. A recent study reported that PZO1 regulated virus translocation within the plant, but its specific role in mechanotransduction was not addressed (23). Here we use genetic tools, electrophysiological methods, and calcium imaging to investigate the role of PZO1 in root mechanosensation.     

Evidence for five divergent thioredoxin h sequences in Arabidopsis thaliana.

Five different clones encoding thioredoxin homologues were isolated from Arabidopsis thaliana cDNA libraries. On the basis of the sequences they encode divergent proteins, but all belong to the cytoplasmic thioredoxins h previously described in higher plants. The five proteins obtained by overexpressing the coding sequences in Escherichia coli present typical thioredoxin activities (NADP(+)-malate dehydrogenase activation and reduction by Arabidopsis thioredoxin reductase) despite the presence of a variant active site, Trp-Cys-Pro-Pro-Cys, in three proteins in place of the canonical Trp-Cys-Gly-Pro-Cys sequence described for thioredoxins in prokaryotes and eukaryotes. Southern blots show that each cDNA is encoded by a single gene but suggest the presence of additional related sequences in the Arabidopsis genome. This very complex diversity of thioredoxins h is probably common to all higher plants, since the Arabidopsis sequences appear to have diverged very early, at the beginning of plant speciation. This diversity allows the transduction of a redox signal into multiple pathways.     

Genetic variation for disease resistance and tolerance among Arabidopsis thaliana accessions

Pathogens can be an important selective agent in plant evolution because they can severely reduce plant fitness and growth. However, the role of pathogen selection on plant evolution depends on the extent of genetic variation for resistance traits and their covariance with host fitness. Although it is usually assumed that resistance traits will covary with plant fitness, this assumption has not been tested rigorously in plant-pathogen interactions. Many plant species are tolerant to herbivores, decoupling the relationship between resistance and fitness. Tolerance to pathogens can reduce selection for resistance and alter the effect of pathogens on plant evolution. In this study, we measured three components of Arabidopsis thaliana resistance (pathogen growth, disease symptoms, and host fitness) to the bacteria Pseudomonas syringae and investigated their covariation to determine the relative importance of resistance and tolerance. We observed extensive quantitative variation in the severity of disease symptoms, the bacterial population size, and the effect of infection on host fitness among 19 accessions of A. thaliana infected with P. syringae. The severity of disease symptoms was strongly and positively correlated with bacterial population size. Although the average fitness of infected plants was smaller than noninfected plants, we found no correlation between the bacterial growth or symptoms expressed by different accessions of A. thaliana and their relative fitness after infection. These results indicate that the accessions studied vary in tolerance to P. syringae, reducing the strength of selection on resistance traits, and that symptoms and bacterial growth are not good predictors of host fitness.     

Integration of responses within and across Arabidopsis natural accessions uncovers loci controlling root systems architecture

Phenotypic plasticity is presumed to be involved in adaptive change toward species diversification. We thus examined how candidate genes underlying natural variation across populations might also mediate plasticity within an individual. Our implementation of an integrative “plasticity space” approach revealed that the root plasticity of a single Arabidopsis accession exposed to distinct environments broadly recapitulates the natural variation “space.” Genome-wide association mapping identified the known gene PHOSPHATE 1 (PHO1) and other genes such as Root System Architecture 1 (RSA1) associated with differences in root allometry, a highly plastic trait capturing the distribution of lateral roots along the primary axis. The response of mutants in the Columbia-0 background suggests their involvement in signaling key modulators of root development including auxin, abscisic acid, and nitrate. Moreover, genotype-by-environment interactions for the PHO1 and RSA1 genes in Columbia-0 phenocopy the root allometry of other natural variants. This finding supports a role for plasticity responses in phenotypic evolution in natural environments.A long-standing debate in evolutionary biology is the relevance of phenotypic plasticity as a mechanism leading to species diversity (1). It has been argued that selection on plasticity responses to environment pressures could underlie fixed phenotypic changes between natural variants (2), providing a potentially rapid mechanism of evolutionary change (3). Thus, we tested the hypothesis that genes that enable a functional response to the environment within a population also underlie adaptive changes across natural variants. Arabidopsis thaliana offers ample opportunity to study genes involved in phenotypic plasticity in response to experimental laboratory perturbations, whereas its natural variants offer the opportunity to study the genetic basis for developmental variation observed in nature. We took a unique approach to integrating results from these two perspectives to uncover the molecular basis underlying individual plasticity and variation among natural variants. We focused on the root architectural system because it shows a high degree of plasticity under diverse environmental conditions (4–9). We used a quantitative phenotyping model to capture and integrate plastic changes in root system architecture in response to a range of experimental treatments within the laboratory reference accession Columbia-0 (Col-0). We next cross-referenced this plasticity space derived for an individual accession (Col-0) to the range of phenotypic differences in root architecture observed across Arabidopsis accessions that represent the extent of natural variation under one condition. This allowed us to identify candidate genes underlying root systems architecture using genome-wide association mapping and to test their role in individual plasticity using mutants.