A mechanosensitive anion channel in Arabidopsis thaliana mesophyll cells

Mechanosensitive ion channels are expected to play important roles in transducing mechanical stimuli into intracellular signals during the development and morphogenesis of higher plants. We have identified a novel mechanosensitive anion channel in the protoplast of Arabidopsis thaliana mesophyll cells by using the patch-clamp technique. The channel in the outside-out patches could be activated by positive pressure in the pipette while negative pressure had no effect. The amphipathic membrane crenator trinitrophenol, which is supposed to preferentially insert in the outer leaflet of the lipid bilayer of the plasma membrane, synergized with mechanical membrane stretch to activate the channel. These results suggest that the channel activation is mediated by a convex curvature of the plasma membrane. Therefore, activation of this channel may play an important role when cell volume is increasing during cell growth or hypo-osmotic challenge, which is accompanied by membrane stretch with increasingly convex curvature.     

Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells

The plant nucleus changes its intracellular position not only upon cell division and cell growth but also in response to environmental stimuli such as light. We found that the nucleus takes different intracellular positions depending on blue light in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in mesophyll cells were positioned at the center of the bottom of cells (dark position). Under blue light at 100 mumol m(-2) s(-1), in contrast, nuclei were located along the anticlinal walls (light position). The nuclear positioning from the dark position to the light position was fully induced within a few hours of blue light illumination, and it was a reversible response. The response was also observed in epidermal cells, which have no chloroplasts, suggesting that the nucleus has the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced specifically by blue light at >50 mumol m(-2) s(-1). Furthermore, the response to blue light was induced in phot1 but not in phot2 and phot1phot2 mutants. Unexpectedly, we also found that nuclei as well as chloroplasts in phot2 and phot1phot2 mutants took unusual intracellular positions under both dark and light conditions. The lack of the response and the unusual positioning of nuclei and chloroplasts in the phot2 mutant were recovered by externally introducing the PHOT2 gene into the mutant. These results indicate that phot2 mediates the blue light-dependent nuclear positioning and the proper positioning of nuclei and chloroplasts. This is the first characterization of light-dependent nuclear positioning in spermatophytes.     

Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana

Summary. In leaf mesophyll cells of transgenic Arabidopsis thaliana plants expressing GFP in the chloroplast, stromules (stroma-filled tubules) with a length of up to 20 μm and a diameter of about 400–600 nm are observed in cells with spaces between the chloroplasts. They appear extremely dynamic, occasionally branched or polymorphic. In order to investigate the effect of temperature on chloroplasts, we have constructed a special temperature-controlled chamber for usage with a light microscope (LM-TCC). This LM-TCC enables presetting of the temperature for investigation directly at the microscope stage with an accuracy of ±0.1 °C in a temperature range of 0 °C to +60 °C. With the LM-TCC a temperature-dependent appearance of chloroplast protrusions has been found. These structures have a considerably smaller length-to-diameter ratio than typical stromules and reach a length of 3–5 μm. At 5–15 °C (low temperatures), almost no chloroplast protrusions are observed, but they appear with increasing temperatures. At 35–45 °C (high temperatures), numerous chloroplast protrusions with a beaklike appearance extend from a single chloroplast. Interaction of stromules with other organelles has also been investigated by transmission electron microscopy. At 20 °C, transverse sections of stromules are frequently observed with a diameter of about 450 nm. A close membrane-to-membrane contact of stromules with the nucleus and mitochondria has been visualised. Golgi stacks and microbodies are found in the spatial vicinity of stromules. At 5 °C, virtually no chloroplast protrusions or stromules are observed. At 35 °C, chloroplast protrusions are present as broader thylakoid-free stroma-filled areas, resulting in an irregular chloroplast appearance. Correspondence and reprints: Department of Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.     

Wounding of Arabidopsis leaves induces indole‐3‐carbinol‐dependent autophagy in roots of Arabidopsis thaliana

In cruciferous plants insect attack or physical damage induce the synthesis of the glucosinolate breakdown product indole‐3‐carbinol, which plays a key role in the defense against attackers. Indole‐3‐carbinol also affects plant growth and development, acting as an auxin antagonist by binding to the TIR1 auxin receptor. Other potential functions of indole‐3‐carbinol and the underlying mechanisms in plant biology are unknown. Here we show that an indole‐3‐carbinol‐dependent signal induces specific autophagy in root cells. Leaf treatment with exogenous indole‐3‐carbinol or leaf‐wounding induced autophagy and inhibited auxin response in the root. This induction is lost in glucosinolate‐defective mutants, indicating that the effect of indole‐3‐carbinol is transported in the plants. Thus, indole‐3‐carbinol is not only a defensive metabolite that repels insects, but is also involved in long‐distance communication regulating growth and development in plants.     

Transient expression in leaf mesophyll protoplasts of Arabidopsis thaliana

Conditions for maximising transient expression of GUS in leaf mesophyll protoplasts of Arabidopsis thaliana ecotype C24 were investigated. It was found that the factors most influencing expression levels, with optimum levels in parenthesis, were plasmid DNA quantity (100 g per 5 × 10⁵ protoplasts), inclusion of carrier DNA (50 g), PEG pH and amount (pH above 6, and total PEG concentration at least 9% w/w) and the topological form of the DNA. Linearised plasmid DNA with long flanking sequences 3 and 5 to the marker gene yielded the highest levels of GUS expression.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - MU methylumbelliferone - PEG polyethylene glycol - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid     

MIDGET connects COP1‐dependent development with endoreduplication in Arabidopsis thaliana

In Arabidopsis thaliana, loss of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) function leads to constitutive photomorphogenesis in the dark associated with inhibition of endoreduplication in the hypocotyl, and a post‐germination growth arrest. MIDGET (MID), a component of the TOPOISOMERASE VI (TOPOVI) complex, is essential for endoreduplication and genome integrity in A. thaliana. Here we show that MID and COP1 interact in vitro and in vivo through the amino terminus of COP1. We further demonstrate that MID supports sub‐nuclear accumulation of COP1. The MID protein is not degraded in a COP1‐dependent fashion in darkness, and the phenotypes of single and double mutants prove that MID is not a target of COP1 but rather a necessary factor for proper COP1 activity with respect to both, control of COP1‐dependent morphogenesis and regulation of endoreduplication. Our data provide evidence for a functional connection between COP1 and the TOPOVI in plants linking COP1‐dependent development with the regulation of endoreduplication.     

Low-temperature-induced transmembrane potential changes in mesophyll cells of Arabidopsis thaliana, Helianthus annuus and Vicia faba

Glass microelectrodes were inserted into mesophyll cells of intact leaves from higher plants: Arabidopsis thaliana, Helianthus annuus and Vicia faba var minor , and transient membrane potential changes were recorded in response to a sudden temperature drop. The cold-induced potential changes were unaffected by an anion channel inhibitor (anthracene-9-carboxylic acid) and potassium channel inhibitor (tetraethyl ammonium ion). Verapamil, a calcium channel inhibitor, caused significant suppression of the cold-induced potential changes. In the presence of calmoduline antagonists (trifluoperazine and N -6-aminohexyl-5-chloro-1-naphtalenesulphonamide), their amplitudes decreased and their durations were prolonged. Neomycin, which suppresses phospholipase C, also caused substantial inhibition of the amplitudes of the cold-induced potential changes. It is concluded that cold-evoked membrane potential changes are due to calcium influxes from both the apoplast and internal stores.     

The mitochondrial complexome of Arabidopsis thaliana

Mitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein–protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot‐gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re‐annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource ( https://complexomemap.de/at_mito_leaves ) to deposit the data and to allow straightforward access and custom data analyses.     

The role of Arabidopsis thaliana RASD1 gene in ABA‐dependent abiotic stress response

• Abiotic stress is one of the key parameters affecting plant productivity. Drought and soil salinity, in particular, challenge plants to activate various response mechanisms to withstand these adverse growth conditions. While the molecular events that take place are complex and to a large extent unclear, the plant hormone abscisic acid (ABA) is considered a major player in mediating the adaptation of plants to stress.
• Here we report the identification of an ABA‐insensitive mutant from Arabidopsis thaliana. A combination of molecular, genetic and physiology approaches were implemented, to characterise the AtRASD1 locus (A BA D ROUGHT 相似文献   

Dismantling of Arabidopsis thaliana mesophyll cell chloroplasts during natural leaf senescence

One of the earliest events in the process of leaf senescence is dismantling of chloroplasts. Mesophyll cell chloroplasts from rosette leaves were studied in Arabidopsis thaliana undergoing natural senescence. The number of chloroplasts decreased by only 17% in fully yellow leaves, and chloroplasts were found to undergo progressive photosynthetic and ultrastructural changes as senescence proceeded. In ultrastructural studies, an intact tonoplast could not be visualized, thus, a 35S-GFP::δ-TIP line with a GFP-labeled tonoplast was used to demonstrate that chloroplasts remain outside of the tonoplast even at late stages of senescence. Chloroplast DNA was measured by real-time PCR at four different chloroplast loci, and a fourfold decrease in chloroplast DNA per chloroplast was noted in yellow senescent leaves when compared to green leaves from plants of the same age. Although chloroplast DNA did decrease, the chloroplast/nuclear gene copy ratio was still 31:1 in yellow leaves. Interestingly, mRNA levels for the four loci differed: psbA and ndhB mRNAs remained abundant late into senescence, while rpoC1 and rbcL mRNAs decreased in parallel to chloroplast DNA. Together, these data demonstrate that, during senescence, chloroplasts remain outside of the vacuole as distinct organelles while the thylakoid membranes are dismantled internally. As thylakoids were dismantled, Rubisco large subunit, Lhcb1, and chloroplast DNA levels declined, but variable levels of mRNA persisted.     

Genetic architecture of mitochondrial editing in Arabidopsis thaliana

We have analyzed the mitochondrial editing behavior of two Arabidopsis thaliana accessions, Landsberg erecta (Ler) and Columbia (Col). A survey of 362 C-to-U editing sites in 33 mitochondrial genes was conducted on RNA extracted from rosette leaves. We detected 67 new editing events in A. thaliana rosette leaves that had not been observed in a prior study of mitochondrial editing in suspension cultures. Furthermore, 37 of the 441 C-to-U editing events reported in A. thaliana suspension cultures were not observed in rosette leaves. Forty editing sites that are polymorphic in extent of editing were detected between Col and Ler. Silent editing sites, which do not change the encoded amino acid, were found in a large excess compared to nonsilent sites among the editing events that differed between accessions and between tissue types. Dominance relationships were assessed for 15 of the most polymorphic sites by evaluating the editing values of the reciprocal hybrids. Dominance is more common in nonsilent sites than in silent sites, while additivity was observed only in silent sites. A maternal effect was detected for 8 sites. QTL mapping with recombinant inbred lines detected 12 major QTL for 11 of the 13 editing traits analyzed, demonstrating that efficiency of editing of individual mitochondrial C targets is generally governed by a major factor.     

Trehalose phosphate synthase 5‐dependent trehalose metabolism modulates basal defense responses in Arabidopsis thaliana

Despite the recent discovery that trehalose synthesis is important for plant development and abiotic stress tolerance, the effects of trehalose on biotic stress responses remain relatively unknown. In this study, we demonstrate that TREHALOSE PHOSPHATE SYNTHASE 5 (TPS5)-dependent trehalose metabolism regulates Arabidopsis thaliana defenses against pathogens (necrotrophic Botrytis cinerea and biotrophic Pseudomonas syringae). Pathogen infection increased trehalose levels and upregulated TPS5 expression. Application of exogenous trehalose significantly improved plant defenses against B. cinerea, but increased the susceptibility of plants to P. syringae. We demonstrate that elevated trehalose biosynthesis, in transgenic plants over-expressing TPS5, also increased the susceptibility to P. syringae, but decreased the disease symptoms caused by B. cinerea. The knockout of TPS5 prevented the accumulation of trehalose and enhanced defense responses against P. syringae. Additionally, we observed that a TPS5-interacting protein (multiprotein bridging factor 1c) was required for induced expression of TPS5 during pathogen infections. Furthermore, we show that trehalose promotes P. syringae growth and disease development, via a mechanism involving suppression of the plant defense gene, Pathogenesis-Related Protein 1. These findings provide insight into the function of TPS5-dependent trehalose metabolism in plant basal defense responses.     

Polyploidy‐associated genomic instability in Arabidopsis thaliana

Formation of polyploid organisms by fertilization of unreduced gametes in meiotic mutants is believed to be a common phenomenon in species evolution. However, not well understood is how species in nature generally exist as haploid and diploid organisms in a long evolutionary time while polyploidization must have repeatedly occurred via meiotic mutations. Here, we show that the ploidy increased for two consecutive generations due to unreduced but viable gametes in the Arabidopsis cyclin a1;2‐2 (also named tardy asynchronous meiosis‐2) mutant, but the resultant octaploid plants produced progeny of either the same or reduced ploidy via genomic reductions during meiosis and pollen mitosis. Ploidy reductions through sexual reproduction were also observed in independently generated artificial octaploid and hexaploid Arabidopsis plants. These results demonstrate that octaploid is likely the maximal ploidy produced through sexual reproduction in Arabidopsis. The polyploidy‐associated genomic instability may be a general phenomenon that constrains ploidy levels in species evolution. genesis 48:254–263, 2010. © 2010 Wiley‐Liss, Inc.     

Cyclin‐dependent kinase‐activating kinases CDKD;1 and CDKD;3 are essential for preserving mitotic activity in Arabidopsis thaliana

For the full activation of cyclin‐dependent kinases (CDKs), not only cyclin binding but also CDK phosphorylation is required. This activating phosphorylation is mediated by CDK‐activating kinases (CAKs). Arabidopsis has four genes showing similarity to vertebrate‐type CAKs, three CDKDs (CDKD;1‐CDKD;3) and one CDKF (CDKF;1). We previously found that the cdkf;1 mutant is defective in post‐embryonic development, even though the kinase activities of core CDKs remain unchanged relative to the wild type. This raised a question about the involvement of CDKDs in CDK activation in planta. Here we report that the cdkd;1 cdkd;3 double mutant showed gametophytic lethality. Most cdkd;1‐1 cdkd;3‐1 pollen grains were defective in pollen mitosis I and II, producing one‐cell or two‐cell pollen grains that lacked fertilization ability. We also found that the double knock‐out of CDKD;1 and CDKD;3 caused arrest and/or delay in the progression of female gametogenesis at multiple steps. Our genetic analyses revealed that the functions of CDKF;1 and CDKD;1 or CDKD;3 do not overlap, either during gametophyte and embryo development or in post‐embryonic development. Consistent with these analyses, CDKF;1 expression in the cdkd;1‐1 cdkd;3‐1 mutant could not rescue the gametophytic lethality. These results suggest that, in Arabidopsis, CDKD;1 and CDKD;3 function as CAKs controlling mitosis, whereas CDKF;1 plays a distinct role, mainly in post‐embryonic development. We propose that CDKD;1 and CDKD;3 phosphorylate and activate all core CDKs, CDKA, CDKB1 and CDKB2, thereby governing cell cycle progression throughout plant development.     

Plasmodesmata in Arabidopsis thaliana suspension cells

Summary. A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs). The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana. This material has the advantages of homogeneity, quantity, and ease of disruption. Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth. Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells. Calreticulin and the chemical stain 3,3-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy. Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs. Hence, clean cell walls from A. thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.Correspondence and reprints: John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.