New insights into the functional roles of reactive oxygen species during embryo sac development and fertilization in Arabidopsis thaliana

Previously considered as toxic by-products of aerobic metabolism, reactive oxygen species (ROS) are emerging as essential signaling molecules in eukaryotes. Recent evidence showed that maintenance of ROS homeostasis during female gametophyte development is crucial for embryo sac patterning and fertilization. Although ROS are exclusively detected in the central cell of mature embryo sacs, the study of mutants deficient in ROS homeostasis suggests that controlled oxidative bursts might take place earlier during gametophyte development. Also, a ROS burst that depends on pollination takes place inside the embryo sac. This oxidative response might be required for pollen tube growth arrest and for sperm cell release. In this mini-review, we will focus on new insights into the role of ROS during female gametophyte development and fertilization. Special focus will be made on the mitochondrial Mn-Superoxide dismutase (MSD1), which has been recently reported to be essential for maintaining ROS homeostasis during embryo sac formation.     

Arabidopsis thaliana as a model for functional nectary analysis

Nectaries and nectar have received much research attention for well over 200 years due to their central roles in plant–pollinator interactions. Despite this, only a few genes have demonstrated impacts on nectary development, and none have been reported to mediate de novo nectar production. This scarcity of information is largely due to the lack of a model that combines sizeable nectaries, and high levels of nectar production, along with suitable genomics resources. For example, even though Arabidopsis thaliana has been useful for developmental studies, it has been largely overlooked as a model for studying nectary function due to the small size of its flowers. However, Arabidopsis nectaries, along with those of related species, are quite operational and can be used to discern molecular mechanisms of nectary form and function. A current understanding of the machinery underlying nectary function in plants is briefly presented, with emphasis placed on the prospects of using Arabidopsis as a model for studying these processes.     

A multigene family of glycosyltransferases in a model plant,Arabidopsis thaliana

Glycosyltransferases transfer sugars from NDP-sugar donors to acceptors. The multigene family of transferases described in this paper typically transfer glucose from UDP-glucose to low-molecular-mass acceptors in the cytosol of plant cells. There are 107 sequences in the genome of Arabidopsis thaliana that contain a consensus, suggesting they belong to this Group 1 multigene family. The family has been analysed phylogenetically, and a functional genomics approach has been applied to explore the relatedness of sequence similarity to catalytic specificity and stereoselectivity. Enzymes belonging to this class of transferases glycosylate a vast array of acceptors, including natural products such as secondary metabolites and hormones, as well as xenobiotics absorbed by the plant, such as herbicides and pesticides. Conjugation to glucose potentially changes the activity of the acceptor molecule and invariably changes its location within the plant cell. Using the genomics approach described, a platform of knowledge has been constructed that will enable an understanding to be gained on the role of these enzymes in cellular homoeostasis, as well as their activity in biotransformations in vitro that require strict regioselectivity of glycosylation.     

Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana

Diversity Arrays Technology (DArT) is a microarray-based DNA marker technique for genome-wide discovery and genotyping of genetic variation. DArT allows simultaneous scoring of hundreds of restriction site based polymorphisms between genotypes and does not require DNA sequence information or site-specific oligonucleotides. This paper demonstrates the potential of DArT for genetic mapping by validating the quality and molecular basis of the markers, using the model plant Arabidopsis thaliana. Restriction fragments from a genomic representation of the ecotype Landsberg erecta (Ler) were amplified by PCR, individualized by cloning and spotted onto glass slides. The arrays were then hybridized with labeled genomic representations of the ecotypes Columbia (Col) and Ler and of individuals from an F₂ population obtained from a Col × Ler cross. The scoring of markers with specialized software was highly reproducible and 107 markers could unambiguously be ordered on a genetic linkage map. The marker order on the genetic linkage map coincided with the order on the DNA sequence map. Sequencing of the Ler markers and alignment with the available Col genome sequence confirmed that the polymorphism in DArT markers is largely a result of restriction site polymorphisms.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Identification of novel families of membrane proteins from the model plant Arabidopsis thaliana

MOTIVATION: The completion of the Arabidopsis genome offers the first opportunity to analyze all of the membrane protein sequences of a plant. The majority of integral membrane proteins including transporters, channels, and pumps contain hydrophobic alpha-helices and can be selected based on TransMembrane Spanning (TMS) domain prediction. By clustering the predicted membrane proteins based on sequence, it is possible to sort the membrane proteins into families of known function, based on experimental evidence or homology, or unknown function. This provides a way to identify target sequences for future functional analysis. RESULTS: An automated approach was used to select potential membrane protein sequences from the set of all predicted proteins and cluster the sequences into related families. The recently completed sequence of Arabidopsis thaliana, a model plant, was analyzed. Of the 25,470 predicted protein sequences 4589 (18%) were identified as containing two or more membrane spanning domains. The membrane protein sequences clustered into 628 distinct families containing 3208 sequences. Of these, 211 families (1764 sequences) either contained proteins of known function or showed homology to proteins of known function in other species. However, 417 families (1444 sequences) contained only sequences with no known function and no homology to proteins of known function. In addition, 1381 sequences did not cluster with any family and no function could be assigned to 1337 of these.     

Characterizing genomic variation of Arabidopsis thaliana: the roles of geography and climate

Arabidopsis thaliana inhabits diverse climates and exhibits varied phenology across its range. Although A. thaliana is an extremely well‐studied model species, the relationship between geography, growing season climate and its genetic variation is poorly characterized. We used redundancy analysis (RDA) to quantify the association of genomic variation [214 051 single nucleotide polymorphisms (SNPs)] with geography and climate among 1003 accessions collected from 447 locations in Eurasia. We identified climate variables most correlated with genomic variation, which may be important selective gradients related to local adaptation across the species range. Climate variation among sites of origin explained slightly more genomic variation than geographical distance. Large‐scale spatial gradients and early spring temperatures explained the most genomic variation, while growing season and summer conditions explained the most after controlling for spatial structure. SNP variation in Scandinavia showed the greatest climate structure among regions, possibly because of relatively consistent phenology and life history of populations in this region. Climate variation explained more variation among nonsynonymous SNPs than expected by chance, suggesting that much of the climatic structure of SNP correlations is due to changes in coding sequence that may underlie local adaptation.     

Arabidopsis thaliana: protocol for plant regeneration from protoplasts

We report a protocol for plant regeneration from leaf protoplasts of Arabidopsis thaliana ecotype Zürich. The protocol has been established in 1988 and has since been in routine use in our laboratory. Whereas recovery of proliferating protoplast-derived clones is routine, the success in plant regeneration from protoplast-derived clones is highly variable. In the hands of one of us (H.K.) average shoot regeneration frequency (% of calli regenerating at least one shoot) was ca. 60% and average plant regeneration frequency (% of calli yielding fertile plants in soil) was ca. 40%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid - BAP benzylaminopurine - 2-iP 2-isopentenylaminopurine     

Human intrinsic factor expressed in the plant Arabidopsis thaliana.

Intrinsic factor (IF) is the gastric protein that promotes the intestinal uptake of vitamin B12. Gastric IF from animal sources is used in diagnostic tests and in vitamin pills. However, administration of animal IF to humans becomes disadvantageous because of possible pathogenic transmission and contamination by other B12 binders. We tested the use of recombinant plants for large-scale production of pathogen-free human recombinant IF. Human IF was successfully expressed in the recombinant plant Arabidopsis thaliana. Extract from fresh plants possessed high B12-binding capacity corresponding to 70 mg IF per 1 kg wet weight. The dried plants still retained 60% of the IF activity. The purified IF preparation consisted of a 50-kDa glycosylated protein with the N-terminal sequence of mature IF. Approximately one-third of the protein was cleaved at the internal site em leader PSNP downward arrow GPGP. The key properties of the preparation obtained were identical to those of native IF: the binding curves of vitamin B12 to recombinant IF and gastric IF were the same, as were those for a B12 analogue cobinamide, which binds to IF with low affinity. The absorbance spectra of the vitamin bound to recombinant IF and gastric IF were alike, as was the interaction of recombinant and native IF with the specific receptor cubilin. The data presented show that recombinant plants have a great potential as a large-scale source of human IF for analytical and therapeutic purposes.     

The roles of autophagy in development and stress responses in Arabidopsis thaliana

Autophagy is a dynamic process that involves the recycling process of the degradation of intracellular materials. Over the past decade, our molecular and physiological understanding of plant autophagy has greatly been increased. Most essential autophagic machineries are conserved from yeast to plants. The roles that autophagy-related genes (ATGs) family play in the lifecycle of the Arabidopsis are proved to be similar to that in mammal. Autophagy is activated during certain stages of development, senescence or in response to starvation, or environmental stress in Arabidopsis. In the progression of autophagy, ATGs act as central signaling regulators and could develop sophisticated mechanisms to survive when plants are suffering unfavorable environments. It will facilitate further understanding of the molecular mechanisms of autophagy in plant. In this review, we will discuss recent advances in our understanding of autophagy in Arabidopsis, areas of controversy, and highlight potential future directions in autophagy research.     

Root K(+) acquisition in plants: the Arabidopsis thaliana model

K(+) is an essential macronutrient required by plants to complete their life cycle. It fulfills important functions and it is widely used as a fertilizer to increase crop production. Thus, the identification of the systems involved in K(+) acquisition by plants has always been a research goal as it may eventually produce molecular tools to enhance crop productivity further. This review is focused on the recent findings on the systems involved in K(+) acquisition. From Epstein's pioneering work >40 years ago, K(+) uptake was considered to consist of a high- and a low-affinity component. The subsequent molecular approaches identified genes encoding K(+) transport systems which could be involved in the first step of K(+) uptake at the plant root. Insights into the regulation of these genes and the proteins that they encode have also been gained in recent studies. A demonstration of the role of the two main K(+) uptake systems at the root, AtHKA5 and AKT1, has been possible with the study of Arabidopsis thaliana T-DNA insertion lines that knock out these genes. AtHAK5 was revealed as the only uptake system at external concentrations <10 μM. Between 10 and 200 μM both AtHAK5 and AKT1 contribute to K(+) acquisition. At external concentrations >500 μM, AtHAK5 is not relevant and AKT1's contribution to K(+) uptake becomes more important. At 10 mM K(+), unidentified systems may provide sufficient K(+) uptake for plant growth.     

Coding sequence divergence between two closely related plant species: Arabidopsis thaliana and Brassica rapa ssp. pekinensis

To characterize the coding-sequence divergence of closely related genomes, we compared DNA sequence divergence between sequences from a Brassica rapa ssp. pekinensis EST library isolated from flower buds and genomic sequences from Arabidopsis thaliana. The specific objectives were (i) to determine the distribution of and relationship between K _a and K _s, (ii) to identify genes with the lowest and highest K _a:K _s values, and (iii) to evaluate how codon usage has diverged between two closely related species. We found that the distribution of K _a:K _s was unimodal, and that substitution rates were more variable at nonsynonymous than synonymous sites, and detected no evidence that K _a and K _s were positively correlated. Several genes had K _a:K _s values equal to or near zero, as expected for genes that have evolved under strong selective constraint. In contrast, there were no genes with K _a:K _s >1 and thus we found no strong evidence that any of the 218 sequences we analyzed have evolved in response to positive selection. We detected a stronger codon bias but a lower frequency of GC at synonymous sites in A. thaliana than B. rapa. Moreover, there has been a shift in the profile of most commonly used synonymous codons since these two species diverged from one another. This shift in codon usage may have been caused by stronger selection acting on codon usage or by a shift in the direction of mutational bias in the B. rapa phylogenetic lineage.     

Preparation and functional characterization of thylakoids from Arabidopsis thaliana

A protocol for the isolation of functional thylakoids from Arabidopsis thaliana leaves was developed. The critical factor in obtaining active, coupled and stable preparation is the inclusion of EDTA and EGTA in the grinding buffer. Preparations were characterized with respect to the whole or partial electron transport chain, ATP/NADPH, ATP/O₂ and PS II/chlorophyll ratios. Sensitivity to a light-chill photoinhibitory treatment was also determined by evaluating the decrease in both maximal photochemical efficiency (Fv/Fm) and in electron transport rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Root growth is affected differently by mechanical wounding in seedlings of the ecological model species Nicotiana attenuata and the molecular model species Arabidopsis thaliana

In the ecological model plant Nicotiana attenuata, leaf wounding or herbivory lead to a reduction of root growth via jasmonic acid (JA) signaling. A single wounding treatment is sufficient to induce this response; multiple wounding does not increase the plant growth reaction. in a recent study, in which JA bursts were elicited in leaves of the molecular model species Arabidopsis thaliana in different ways,¹ we tested whether JA induces the same response there. Root growth reduction was neither induced by foliar application of herbivore oral secretions nor by direct application of methyl jasmonate to leaves. Root growth reduction was observed when leaves were infected with the pathogen Pseudomonas syringae pv. tomato, which persistently induces the JA signaling pathway. Yet, growth analyses of this effect in wild type and JA-signaling mutants showed that it was elicited by the bacterial toxin coronatine which suggests ethylene—but not JA-induced root growth reduction in A. thaliana. Moreover, the growth effects were somewhat masked by a light-induced diurnal decrease of root growth. Overall, we conclude that the reaction of root growth to herbivore-induced JA signaling differs among species, which is related to different ecological defence strategies that have evolved in different species.Key words: coronatine, ethylene, image analysis, phytohormones, Pseudomonas syringae pv. tomato, woundingUpon pathogen or herbivore attack, plants have to meet the decision how much of their resources are invested in growth processes and how much into defense. The ecological model species Nicotiana attenuata increases defence measures and decreases root, but not leaf growth immediately after a single simulated herbivory event.² This reaction is elucidated via jasmonic acid (JA) signaling.³ The intensity of root growth reduction is not amplified when multiple wounding events occur (Fig. 1A). This clearly demonstrates that wounding acts as a signal for the reduction of root growth and that root growth is not reduced due to a lack of growth resources as a consequence of a resource-based trade-off between growth and defence. This hypothesis is further supported by the finding that a surplus of carbohydrates is stored in the root system,⁴ which thereby acts as a safe retreat for future re-growth of the plant after herbivore damage.Open in a separate windowFigure 1Root growth in Nicotiana attenuata and Arabidopsis thaliana seedlings. (A) Root growth dynamics of Nicotiana attenuata seedlings after single and multiple wounding treatments as well as multiple wounding treatments followed by application of oral secretions of Manduca sexta (OS). Wounding treatments were applied at time points 0 h (single treatments) or at the time points 0 h, 2 h and 4 h (multiple treatments). Controls were not treated. (B) Normalized values of velocity of the root tip (v_Tip) of Arabidopsis thaliana seedlings whose roots were exposed to light (control and wounded) and seedlings whose roots were darkened by wrapping aluminium foil around the Petri dish throughout the growth period. Shaded areas indicate the night period. Mean ± SE. N = 4–8.We asked ourselves whether this is a general reaction pattern that is followed in more plant species. To test this, we performed a suite of experiments on the molecular model species Arabidopsis thaliana.¹ Several studies showed that direct application of JA or methyl jasmonate (MeJA), which is commonly used to mimick herbivory-induced signaling, to the cultivation medium decrease root growth of A. thaliana. Yet, in contrast to the situation in N. attenuata, the application of MeJA to leaves did not lead to a decrease in root growth. To exclude the possibility that the MeJA applied to the leaf was not taken up by the plants, we induced plant-internal JA bursts by mechanical wounding and/or application of bacteria. The treatments were performed on Col-0 and Col-6 wild type plants. Additionally, two mutants defective in the JA signaling pathway were used to select for JA-induced effects. coi1-1 (coronatine-insensitive) is known to lack the F-box protein COI1 and shows decreased sensitivity to JA application compared to wild type plants.⁵ The aos mutant, in contrast, is unable to produce JA following mechanical wounding as the biosynthesis of the rate-limiting enzyme allene oxide synthase is blocked.⁶Upon mechanical wounding of two leaves with sterile tweezers, JA concentration in the seedlings increased and root growth decreased rapidly, but only very transiently in all four investigated A. thaliana lines. In contrast to the situation in N. attenuata, root growth in A. thaliana recovered to pre-treatment levels within a few hours (Fig. 1B) and growth was not further decreased upon addition of oral secretions of Spodoptera littoralis larvae. This suggests that the observed short-term growth reduction was caused by hydraulic decrease of the plant growth potential. A slight, but continuous decrease of root growth during the day was noted both in wounded and in control plants that were not completely protected from ambient light in the transparent Petri dishes. When root systems were completely protected from ambient light by shading, root growth was almost steady throughout 24 h (Fig. 1B).In another experimental approach to clarify the connection between JA signaling and root growth reduction, we infected leaves with the avirulent Pseudomonas syringae pv. tomato (Pst) DC3000 avrRpt2 strain. Upon mechanical wounding and application of bacterial suspension in order to facilitate infection, root growth decreased more rapidly than upon mere wounding. In the course of two days after infection, v_Tip was lower in the wild types and the aos mutant suggesting that JA was not the major reason of the decrease of root growth. With Pst DC3000 deficient in coronatine biosynthesis, it was verified that the bacterial toxin was the major reason of the root growth reduction following Pst infection. Using the ethylene reception blocker 1-methyl cyclopropene (1-MCP), ethylene was also figured out to be involved in coronatine-mediated root growth impairment in Arabidopsis. Thus, root growth of Arabidopsis is more sensitive to ethylene than to JA which is very different to observations on N. attenuata.The conclusion has to be drawn that elicitation of JA-bursts in the leaves of A. thaliana does not induce the same root growth reactions as in N. attenuata, although roots of both species react towards MeJA externally applied to the cultivation medium. This in turn demonstrates clearly that the interpretation of the JA signal differs between species. Possibly, this reflects different survival strategies to which the two investigated annual rosette species have evolved. While N. attenuata uses the root as a safe retreat for resources allowing later re-growth after the herbivore threat has passed by, A. thaliana is more successful in its ecological niche if it does not slow down growth in response to herbivory but continues its development as rapidly as possible.     

Phototropin from Chlamydomonas reinhardtii is functional in Arabidopsis thaliana

Phototropin, a plant blue light photoreceptor, mediates important blue light responses such as phototropism, chloroplast positioning and stomatal opening in higher plants. In Arabidopsis thaliana, two phototoropins, phototropin 1 and 2, are known. Recently, in the unicellular green alga, Chlamydomonas reinhardtii, a phototropin homolog was identified. It exhibits photochemical properties similar to those of higher plant phototropins and is involved in multiple steps of the sexual life cycle of Chlamydomonas. Here, we expressed Chlamydomonas phototropin in Arabidopsis to examine whether it is active in a distantly related plant species. The Arabidopsis mutant deficient in both phototropin 1 and 2 was transformed with a vector containing Chlamydomonas phototropin cDNA fused to a cauliflower mosaic virus 35S promoter. The resulting lines were classified into high, medium and low expressers based on RNA gel blot and immunoblot analyses. Typical phototropin responses were restored in high expression lines. These results demonstrate that Chlamydomonas phototropin is functional in higher plants. Hence, the basic mechanism of phototropin action is highly conserved, even though its apparent physiological functions are quite diverse.     

Natural developmental variations in leaf and plant senescence in Arabidopsis thaliana

Leaf senescence is a developmentally regulated process that contributes to nutrient redistribution during reproductive growth and finally leads to tissue death. Manipulating leaf senescence through breeding or genetic engineering may help to improve important agronomic traits, such as crop yield and the storage life of harvested organs. Here, we studied natural variations in the regulation of plant senescence among 16 Arabidopsis thaliana accessions. Chlorophyll content and the proportion of yellow leaves were used as indicator parameters to determine leaf and plant senescence respectively. Our study indicated significant genotype effects on the onset and development of senescence. We selected three late- and five early-senescence accessions for further physiological studies. The relationship between leaf and plant senescence was accession-dependent. There was a significant correlation between plant senescence and the total number of leaves, siliques and plant bolting age. We monitored expression of two senescence marker genes, SAG12 and WRKY53 , to evaluate progression of senescence. Our data revealed that chlorophyll content does not fully reflect leaf age, because even fully green leaves had already commenced senescence at the molecular level. Integrating senescence parameters, such as the proportion of senescent leaves, at the whole plant level provided a better indication of the molecular status of the plant than single leaf senescence parameters.     

Intersection of Small RNA Pathways in Arabidopsis thaliana Sub-Nuclear Domains

In Arabidopsis thaliana, functionally diverse small RNA (smRNA) pathways bring about decreased RNA accumulation of target genes via several different mechanisms. Cytological experiments have suggested that the processing of microRNAs (miRNAs) and heterochromatic small interfering RNAs (hc-siRNAs) occurs within a specific nuclear domain that can present Cajal Body (CB) characteristics. It is unclear whether single or multiple smRNA-related domains are found within the same CB and how specialization of the smRNA pathways is determined within this specific sub-compartment. To ascertain whether nuclear smRNA centers are spatially related, we localized key proteins required for siRNA or miRNA biogenesis by immunofluorescence analysis. The intranuclear distribution of the proteins revealed that hc-siRNA, miRNA and trans-acting siRNA (ta-siRNA) pathway proteins accumulate and colocalize within a sub-nuclear structure in the nucleolar periphery. Furthermore, colocalization of miRNA- and siRNA-pathway members with CB markers, and reduced wild-type localization patterns in CB mutants indicates that proper nuclear localization of these proteins requires CB integrity. We hypothesize that these nuclear domains could be important for RNA silencing and may partially explain the functional redundancies and interactions among components of the same protein family. The CB may be the place in the nucleus where Dicer-generated smRNA precursors are processed and assigned to a specific pathway, and where storage, recycling or assembly of RNA interference components takes place.     

Redox enzymes in the plant plasma membrane and their possible roles

Purified plasma membrane (PM) vesicles from higher plants contain redox proteins with low‐molecular‐mass prosthetic groups such as flavins (both FMN and FAD), hemes, metals (Cu, Fe and Mn), thiol groups and possibly naphthoquinone (vitamin K₁), all of which are likely to participate in redox processes. A few enzymes have already been identified: Monodehydroascorbate reductase (EC 1.6.5.4) is firmly bound to the cytosolic surface of the PM where it might be involved in keeping both cytosolic and, together with a b‐type cytochrome, apoplastic ascorbate reduced. A malate dehydrogenase (EC 1.1.1.37) is localized on the inner side of the PM. Several NAD(P)H‐quinone oxidoreductases have been purified from the cytocolic surface of the PM, but their function is still unknown. Different forms of nitrate reductase (EC 1.6.6.1–3) are found attached to, as well as anchored in, the PM where they may act as a nitrate sensor and/or contribute to blue‐light perception, although both functions are speculative. Ferric‐chelate‐reducing enzymes (EC 1.6.99.13) are localized and partially characterized on the inner surface of the PM but they may participate only in the reduction of ferric‐chelates in the cytosol. Very recently a ferric‐chelate‐reducing enzyme containing binding sites for FAD, NADPH and hemes has been identified and suggested to be a trans‐PM protein. This enzyme is involved in the reduction of apoplastic iron prior to uptake of Fe²⁺ and is induced by iron deficiency. The presence of an NADPH oxidase, similar to the so‐called respiratory burst oxidase in mammals, is still an open question. An auxin‐stimulated and cyanide‐insensitive NADH oxidase (possibly a protein disulphide reductase) has been characterized but its identity is still awaiting independent confirmation. Finally, the only trans‐PM redox protein which has been partially purified from plant PM so far is a high‐potential and ascorbate‐reducible b‐type cytochrome. In co‐operation with vitamin K₁ and an NAD(P)H‐quinone oxidoreductase, it may participate in trans‐PM electron transport.