Plant Kinesin Repertoires Expand with New Domain Architecture and Contract with the Loss of Flagella.

Kinesins are eukaryotic microtubule motor proteins subdivided into conserved families with distinct functional roles. While many kinesin families are widespread in eukaryotes, each organismal lineage maintains a unique kinesin repertoire composed of many families with distinct numbers of genes. Previous genomic surveys indicated that land plant kinesin repertoires differ markedly from other eukaryotes. To determine when repertoires diverged during plant evolution, we performed robust phylogenomic analyses of kinesins in 24 representative plants, two algae, two animals, and one yeast. These analyses show that kinesin repertoires expand and contract coincident with major shifts in the biology of algae and land plants. One kinesin family and five subfamilies, each defined by unique domain architectures, emerged in the green algae. Four of those kinesin groups expanded in ancestors of modern land plants, while six other kinesin groups were lost in the ancestors of pollen-bearing plants. Expansions of different kinesin families and subfamilies occurred in moss and angiosperm lineages. Other kinesin families remained stable and did not expand throughout plant evolution. Collectively these data support a radiation of kinesin domain architectures in algae followed by differential positive and negative selection on kinesins families and subfamilies in different lineages of land plants.

Evolution of Eukaryal and Archaeal Pseudouridine Synthase Pus10.

In archaea, pseudouridine (Ψ) synthase Pus10 modifies uridine (U) to Ψ at positions 54 and 55 of tRNA. In contrast, Pus10 is not found in bacteria, where modifications at those two positions are carried out by TrmA (U54 to m5U54) and TruB (U55 to Ψ55). Many eukaryotes have an apparent redundancy; their genomes contain orthologs of archaeal Pus10 and bacterial TrmA and TruB. Although eukaryal Pus10 genes share a conserved catalytic domain with archaeal Pus10 genes, their biological roles are not clear for the two reasons. First, experimental evidence suggests that human Pus10 participates in apoptosis induced by the tumor necrosis factor-related apoptosis-inducing ligand. Whether the function of human Pus10 is in place or in addition to of Ψ synthesis in tRNA is unknown. Second, Pus10 is found in earlier evolutionary branches of fungi (such as chytrid Batrachochytrium) but is absent in all dikaryon fungi surveyed (Ascomycetes and Basidiomycetes). We did a comprehensive analysis of sequenced genomes and found that orthologs of Pus10, TrmA, and TruB were present in all the animals, plants, and protozoa surveyed. This indicates that the common eukaryotic ancestor possesses all the three genes. Next, we examined 116 archaeal and eukaryotic Pus10 protein sequences to find that Pus10 existed as a single copy gene in all the surveyed genomes despite ancestral whole genome duplications had occurred. This indicates a possible deleterious gene dosage effect. Our results suggest that functional redundancy result in gene loss or neofunctionalization in different evolutionary lineages.

Predicted protein-protein interactions in the moss Physcomitrella patens: a new bioinformatic resource.

BACKGROUND: Physcomitrella patens, a haploid dominant plant, is fast becoming a useful molecular genetics and bioinformatics tool due to its key phylogenetic position as a bryophyte in the post-genomic era. Genome sequences from select reference species were compared bioinformatically to Physcomitrella patens using reciprocal blasts with the InParanoid software package. A reference protein interaction database assembled using MySQL by compiling BioGrid, BIND, DIP, and Intact databases was queried for moss orthologs existing for both interacting partners. This method has been used to successfully predict interactions for a number of angiosperm plants. RESULTS: The first predicted protein-protein interactome for a bryophyte based on the interolog method contains 67,740 unique interactions from 5,695 different Physcomitrella patens proteins. Most conserved interactions among proteins were those associated with metabolic processes. Over-represented Gene Ontology categories are reported here. CONCLUSION: Addition of moss, a plant representative 200 million years diverged from angiosperms to interactomic research greatly expands the possibility of conducting comparative analyses giving tremendous insight into network evolution of land plants. This work helps demonstrate the utility of "guilt-by-association" models for predicting protein interactions, providing provisional roadmaps that can be explored using experimental approaches. Included with this dataset is a method for characterizing subnetworks and investigating specific processes, such as the Calvin-Benson-Bassham cycle.

Role of forefinger and thumb loops in production of Ψ54 and Ψ55 in tRNAs by archaeal Pus10.

Pseudouridines (Ψ) are found in structurally and functionally important regions of RNAs. Six families of Ψ synthases, TruA, TruB, TruD, RsuA, RluA, and Pus10 have been identified. Pus10 is present in Archaea and Eukarya. While most archaeal Pus10 produce both tRNA Ψ54 and Ψ55, some produce only Ψ55. Interestingly, human PUS10 has been implicated in apoptosis and Crohn's and Celiac diseases. Homology models of archaeal Pus10 proteins based on the crystal structure of human PUS10 reveal that there are subtle structural differences in all of these Pus10 proteins. These observations suggest that structural changes in homologous proteins may lead to loss, gain, or change of their functions, warranting the need to study the structure-function relationship of these proteins. Using comparison of structural models and a series of mutations, we identified forefinger loop (reminiscent of that of RluA) and an Arg and a Tyr residue of archaeal Pus10 as critical determinants for its Ψ54, but not for its Ψ55 activity. We also found that a Leu residue, in addition to the catalytic Asp, is essential for both activities. Since forefinger loop is needed for both rRNA and tRNA Ψ synthase activities of RluA, but only for tRNA Ψ54 activity of Pus10, archaeal Pus10 proteins must use a different mechanism of recognition for Ψ55 activity. We propose that archaeal Pus10 uses two distinct mechanisms for substrate uridine recognition and binding. However, since we did not observe any mutation that affected only Ψ55 activity, both mechanisms for archaeal Pus10 activities must share some common features.

A common structural blueprint for plant UDP-sugar-producing pyrophosphorylases.

Plant pyrophosphorylases that are capable of producing UDP-sugars, key precursors for glycosylation reactions, include UDP-glucose pyrophosphorylases (A- and B-type), UDP-sugar pyrophosphorylase and UDP-N-acetylglucosamine pyrophosphorylase. Although not sharing significant homology at the amino acid sequence level, the proteins share a common structural blueprint. Their structures are characterized by the presence of the Rossmann fold in the central (catalytic) domain linked to enzyme-specific N-terminal and C-terminal domains, which may play regulatory functions. Molecular mobility between these domains plays an important role in substrate binding and catalysis. Evolutionary relationships and the role of (de)oligomerization as a regulatory mechanism are discussed.

The nematode resistance allele at the rhg1 locus alters the proteome and primary metabolism of soybean roots.

Heterodera glycines, the soybean cyst nematode (SCN), causes the most damaging chronic disease of soybean (Glycine max). Host resistance requires the resistance allele at rhg1. Resistance destroys the giant cells created in the plant's roots by the nematodes about 24 to 48 h after commencement of feeding. In addition, 4 to 8 d later, a systemic acquired resistance develops that discourages later infestations. The molecular mechanisms that control the rhg1-mediated resistance response appear to be multigenic and complex, as judged by transcript abundance changes, even in near isogenic lines (NILs). This study aimed to focus on key posttranscriptional changes by identifying proteins and metabolites that were increased in abundance in both resistant and susceptible NILs. Comparisons were made among NILs 10 d after SCN infestation and without SCN infestation. Two-dimensional gel electrophoresis resolved more than 1,000 protein spots on each gel. Only 30 protein spots with a significant (P < 0.05) difference in abundance of 1.5-fold or more were found among the four treatments. The proteins in these spots were picked, trypsin digested, and analyzed using quadrupole time-of-flight tandem mass spectrometry. Protein identifications could be made for 24 of the 30 spots. Four spots contained two proteins, so that 28 distinct proteins were identified. The proteins were grouped into six functional categories. Metabolite analysis by gas chromatography-mass spectrometry identified 131 metabolites, among which 58 were altered by one or more treatment; 28 were involved in primary metabolism. Taken together, the data showed that 17 pathways were altered by the rhg1 alleles. Pathways altered were associated with systemic acquired resistance-like responses, including xenobiotic, phytoalexin, ascorbate, and inositol metabolism, as well as primary metabolisms like amino acid synthesis and glycolysis. The pathways impacted by the rhg1 allelic state and SCN infestation agreed with transcript abundance analyses but identified a smaller set of key proteins. Six of the proteins lay within the same small region of the interactome identifying a key set of 159 interacting proteins involved in transcriptional control, nuclear localization, and protein degradation. Finally, two proteins (glucose-6-phosphate isomerase [EC 5.3.1.9] and isoflavone reductase [EC 1.3.1.45]) and two metabolites (maltose and an unknown) differed in resistant and susceptible NILs without SCN infestation and may form the basis of a new assay for the selection of resistance to SCN in soybean.

Use of Dual RNA-seq for Systems Biology Analysis of Zea mays and Aspergillus flavus Interaction.

The interaction between Aspergillus flavus and Zea mays is complex, and the identification of plant genes and pathways conferring resistance to the fungus has been challenging. Therefore, the authors undertook a systems biology approach involving dual RNA-seq to determine the simultaneous response from the host and the pathogen. What was dramatically highlighted in the analysis is the uniformity in the development patterns of gene expression of the host and the pathogen during infection. This led to the development of a "stage of infection index" that was subsequently used to categorize the samples before down-stream system biology analysis. Additionally, we were able to ascertain that key maize genes in pathways such as the jasmonate, ethylene and ROS pathways, were up-regulated in the study. The stage of infection index used for the transcriptomic analysis revealed that A. flavus produces a relatively limited number of transcripts during the early stages (0 to 12 h) of infection. At later stages, in A. flavus, transcripts and pathways involved in endosomal transport, aflatoxin production, and carbohydrate metabolism were up-regulated. Multiple WRKY genes targeting the activation of the resistance pathways (i.e., jasmonate, phenylpropanoid, and ethylene) were detected using causal inference analysis. This analysis also revealed, for the first time, the activation of Z. mays resistance genes influencing the expression of specific A. flavus genes. Our results show that A. flavus seems to be reacting to a hostile environment resulting from the activation of resistance pathways in Z. mays. This study revealed the dynamic nature of the interaction between the two organisms.

The word landscape of the non-coding segments of the Arabidopsis thaliana genome.

BACKGROUND: Genome sequences can be conceptualized as arrangements of motifs or words. The frequencies and positional distributions of these words within particular non-coding genomic segments provide important insights into how the words function in processes such as mRNA stability and regulation of gene expression. RESULTS: Using an enumerative word discovery approach, we investigated the frequencies and positional distributions of all 65,536 different 8-letter words in the genome of Arabidopsis thaliana. Focusing on promoter regions, introns, and 3' and 5' untranslated regions (3'UTRs and 5'UTRs), we compared word frequencies in these segments to genome-wide frequencies. The statistically interesting words in each segment were clustered with similar words to generate motif logos. We investigated whether words were clustered at particular locations or were distributed randomly within each genomic segment, and we classified the words using gene expression information from public repositories. Finally, we investigated whether particular sets of words appeared together more frequently than others. CONCLUSION: Our studies provide a detailed view of the word composition of several segments of the non-coding portion of the Arabidopsis genome. Each segment contains a unique word-based signature. The respective signatures consist of the sets of enriched words, 'unwords', and word pairs within a segment, as well as the preferential locations and functional classifications for the signature words. Additionally, the positional distributions of enriched words within the segments highlight possible functional elements, and the co-associations of words in promoter regions likely represent the formation of higher order regulatory modules. This work is an important step toward fully cataloguing the functional elements of the Arabidopsis genome.

UDP-glucose pyrophosphorylase is not rate limiting, but is essential in Arabidopsis.

UDP-glucose pyrophosphorylase (UGPase) produces UDP-glucose which is essential for sucrose and polysaccharide synthesis. Using Arabidopsis, we demonstrated that two UGPase genes (UGP1 and UGP2) are differentially expressed in a variety of organs, with UGP1 being pre-dominant. Co-expression analyses of UGP genes suggest that UGP1 is closely co-regulated with carbohydrate metabolism genes, late embryogenesis and seed loading, while UGP2 is co-regulated with stress response genes, fertilized flowers and photosynthetic genes. We have used Arabidopsis mutants for the UGP genes to characterize the role of both genes. The UGPase activity/protein was reduced by 70, 10 and 85% in ugp1, ugp2 and ugp1/ugp2 double mutant (DK) plants, respectively. A decrease in UGPase activity/protein was accompanied by an increase in expression of USP, a gene for UDP-sugar pyrophosphorylase, suggesting a compensatory mechanism. Generally, the mutants had no effects on soluble sugar/starch content (except in certain cases for DK plants), and there were no differences in cell wall composition/content between the wild type and the mutants. On the other hand, DK plants had greater hypocotyl and root lengths. When grown in the field, the mutants had as much as a 50% decrease in the number of seeds produced (consistent with a substantial decrease in field fitness), suggesting that they would be outcompeted in the field in a few generations. Overall, the data suggest that UGPase is not rate limiting for sucrose/starch and cell wall synthesis, but that it is essential in Arabidopsis.

Novel localization of callose in the spores of Physcomitrella patens and phylogenomics of the callose synthase gene family.

BACKGROUND AND AIMS: Callose involvement in spore development is a plesiomorphic feature of land plants. Correlated light, fluorescence and immuno-electron microscopy was conducted on the developing spores of Physcomitrella patens to probe for callose. Using a bioinformatic approach, the callose synthase (PpCalS) genes were annotated and PpCalS and AtCalS gene families compared, testing the hypothesis that an exine development orthologue is present in P. patens based on deduced polypeptide similarity with AtCalS5, a known exine development gene. METHODS: Spores were stained with aniline blue fluorescent dye. Capsules were prepared for immuno-light and immuno-electron microscopy by gold labelling callose epitopes with monoclonal antibody. BLAST searches were conducted using the AtCalS5 sequence as a query against the P. patens genome. Phylogenomic analysis of the CalS gene family was conducted using PAUP (v.4.1b10). KEY RESULTS: Callose is briefly present in the aperture of developing P. patens spores. The PpCalS gene family consists of 12 copies that fall into three distinct clades with AtCalS genes. PpCalS5 is an orthologue to AtCalS5 with highly conserved domains and 64 % similarity of their deduced polypeptides. CONCLUSIONS: This is the first study to identify the presence of callose in moss spores. AtCalS5 was previously shown to be involved in pollen exine development, thus making PpCalS5 a suspect gene involved in moss spore exine development.

Genome-Wide Computational Identification of Biologically Significant Cis-Regulatory Elements and Associated Transcription Factors from Rice.

The interactions between transcription factors (TFs) and cis-acting regulatory elements (CREs) provide crucial information on the regulation of gene expression. The determination of TF-binding sites and CREs experimentally is costly and time intensive. An in silico identification and annotation of TFs, and the prediction of CREs from rice are made possible by the availability of whole genome sequence and transcriptome data. In this study, we tested the applicability of two algorithms developed for other model systems for the identification of biologically significant CREs of co-expressed genes from rice. CREs were identified from the DNA sequences located upstream from the transcription start sites, untranslated regions (UTRs), and introns, and downstream from the translational stop codons of co-expressed genes. The biologically significance of each CRE was determined by correlating their absence and presence in each gene with that gene's expression profile using a meta-database constructed from 50 rice microarray data sets. The reliability of these methods in the predictions of CREs and their corresponding TFs was supported by previous wet lab experimental data and a literature review. New CREs corresponding to abiotic stresses, biotic stresses, specific tissues, and developmental stages were identified from rice, revealing new pieces of information for future experimental testing. The effectiveness of some-but not all-CREs was found to be affected by copy number, position, and orientation. The corresponding TFs that were most likely correlated with each CRE were also identified. These findings not only contribute to the prioritization of candidates for further analysis, the information also contributes to the understanding of the gene regulatory network.

Impact of chloroplastic- and extracellular-sourced ROS on high light-responsive gene expression in Arabidopsis.

The expression of 28 high light (HL)-responsive genes of Arabidopsis was analysed in response to environmental and physiological factors known to influence the expression of the HL-responsive gene, ASCORBATE PEROXIDASE2 (APX2). Most (81%) of the HL-responsive genes, including APX2, required photosynthetic electron transport for their expression, and were responsive to abscisic acid (ABA; 68%), strengthening the impression that these two signals are crucial in the expression of HL-responsive genes. Further, from the use of mutants altered in reactive oxygen species (ROS) metabolism, it was shown that 61% of these genes, including APX2, may be responsive to chloroplast-sourced ROS. In contrast, apoplastic/plasma membrane-sourced H2O2, in part directed by the respiratory burst NADPH oxidases AtrbohD and AtrbohF, was shown to be important only for APX2 expression. APX2 expression in leaves is limited to bundle sheath parenchyma; however, for the other genes in this study, information on their tissue specificity of expression is sparse. An analysis of expression in petioles, enriched for bundle sheath tissue compared with distal leaf blade, in HL and control leaves showed that 25% of them had >10-fold higher expression in the petiole than in the leaf blade. However, this did not mean that these petiole expression genes followed a pattern of regulation observed for APX2.

Differential tissue/organ-dependent expression of two sucrose- and cold-responsive genes for UDP-glucose pyrophosphorylase in Populus.

Plant UDP-glucose (UDPG) pyrophosphorylase (UGPase) is involved in the production/metabolism of UDPG, a key metabolite for sucrose and cell wall biosynthesis. Two highly similar cDNAs (UGP1 and UGP2) corresponding to UGPase were isolated from cDNA libraries of hybrid aspen (Populus tremula x tremuloides). Expression of both UGPs, as studied by DNA microarrays and EST abundance, was compared to that of three sucrose synthase genes (SUS1-3), also involved in UDPG synthesis. Generally, the UGPs had lower expression than SUS1 and SUS2 genes (especially in tension wood and cambium), with the notable exception of leaves, primary roots and flowers. Based on real-time quantitative PCR, UGP1 in root xylem, leaves and male flowers was by far the predominant transcript, while in other tissues both UGP1 and UGP2 had comparable expression. In leaves, the UGP1 gene, but not UGP2, was upregulated by light and short-term sucrose feeding. Cold treatment led to dramatic organ-specific changes in relative expression of both genes, with UGP2 being upregulated either transiently (leaves), long-term (stems) or not at all (roots), whereas UGP1 was cold-upregulated in all organs. Individual or overall UGP expression patterns only weakly correlated with UGPase activity/protein; however, UGPase activity and protein were correlated in all tissues/conditions. The data suggest that UGPs are differentially expressed at the tissue level and in response to metabolic feedback (sucrose) and cold stress, and point to a tight posttranscriptional/translational control and, possibly, distinct roles for those genes.

Image-Based Analysis to Dissect Vertical Distribution and Horizontal Asymmetry of Conspecific Root System Interactions in Response to Planting Densities, Nutrients and Root Exudates in Arabidopsis thaliana.

Intraspecific competition is an important plant interaction that has been studied extensively aboveground, but less so belowground, due to the difficulties in accessing the root system experimentally. Recent in vivo and in situ automatic imaging advances help understand root system architecture. In this study, a portable imaging platform and a scalable transplant technique were applied to test intraspecific competition in Arabidopsis thaliana. A single green fluorescent protein labeled plant was placed in the center of a grid of different planting densities of neighboring unlabeled plants or empty spaces, into which different treatments were made to the media. The root system of the central plant showed changes in the vertical distribution with increasing neighbor density, becoming more positively kurtotic, and developing an increasing negative skew with time. Horizontal root distribution was initially asymmetric, but became more evenly circular with time, and mean direction was not affected by the presence of adjacent empty spaces as initially hypothesized. To date, this is the first study to analyze the patterns of both vertical and horizontal growth in conspecific root systems. We present a portable imaging platform with simplicity, accessibility, and scalability, to capture the dynamic interactions of plant root systems.

The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination.

Aspergillus flavus is an opportunistic plant pathogen that colonizes and produces the toxic and carcinogenic secondary metabolites, aflatoxins, in oil-rich crops such as maize (Zea mays ssp. mays L.). Pathogenesis-related (PR) proteins serve as an important defense mechanism against invading pathogens by conferring systemic acquired resistance in plants. Among these, production of the PR maize seed protein, ZmPRms (AC205274.3_FG001), has been speculated to be involved in resistance to infection by A. flavus and other pathogens. To better understand the relative contribution of ZmPRms to A. flavus resistance and aflatoxin production, a seed-specific RNA interference (RNAi)-based gene silencing approach was used to develop transgenic maize lines expressing hairpin RNAs to target ZmPRms. Downregulation of ZmPRms in transgenic kernels resulted in a ∼250-350% increase in A. flavus infection accompanied by a ∼4.5-7.5-fold higher accumulation of aflatoxins than control plants. Gene co-expression network analysis of RNA-seq data during the A. flavus-maize interaction identified ZmPRms as a network hub possibly responsible for regulating several downstream candidate genes associated with disease resistance and other biochemical functions. Expression analysis of these candidate genes in the ZmPRms-RNAi lines demonstrated downregulation (vs. control) of a majority of these ZmPRms-regulated genes during A. flavus infection. These results are consistent with a key role of ZmPRms in resistance to A. flavus infection and aflatoxin accumulation in maize kernels.

A Network Approach of Gene Co-expression in the Zea mays/Aspergillus flavus Pathosystem to Map Host/Pathogen Interaction Pathways.

A gene co-expression network (GEN) was generated using a dual RNA-seq study with the fungal pathogen Aspergillus flavus and its plant host Zea mays during the initial 3 days of infection. The analysis deciphered novel pathways and mapped genes of interest in both organisms during the infection. This network revealed a high degree of connectivity in many of the previously recognized pathways in Z. mays such as jasmonic acid, ethylene, and reactive oxygen species (ROS). For the pathogen A. flavus, a link between aflatoxin production and vesicular transport was identified within the network. There was significant interspecies correlation of expression between Z. mays and A. flavus for a subset of 104 Z. mays, and 1942 A. flavus genes. This resulted in an interspecies subnetwork enriched in multiple Z. mays genes involved in the production of ROS. In addition to the ROS from Z. mays, there was enrichment in the vesicular transport pathways and the aflatoxin pathway for A. flavus. Included in these genes, a key aflatoxin cluster regulator, AflS, was found to be co-regulated with multiple Z. mays ROS producing genes within the network, suggesting AflS may be monitoring host ROS levels. The entire GEN for both host and pathogen, and the subset of interspecies correlations, is presented as a tool for hypothesis generation and discovery for events in the early stages of fungal infection of Z. mays by A. flavus.

A predicted protein interactome identifies conserved global networks and disease resistance subnetworks in maize.

Interactomes are genome-wide roadmaps of protein-protein interactions. They have been produced for humans, yeast, the fruit fly, and Arabidopsis thaliana and have become invaluable tools for generating and testing hypotheses. A predicted interactome for Zea mays (PiZeaM) is presented here as an aid to the research community for this valuable crop species. PiZeaM was built using a proven method of interologs (interacting orthologs) that were identified using both one-to-one and many-to-many orthology between genomes of maize and reference species. Where both maize orthologs occurred for an experimentally determined interaction in the reference species, we predicted a likely interaction in maize. A total of 49,026 unique interactions for 6004 maize proteins were predicted. These interactions are enriched for processes that are evolutionarily conserved, but include many otherwise poorly annotated proteins in maize. The predicted maize interactions were further analyzed by comparing annotation of interacting proteins, including different layers of ontology. A map of pairwise gene co-expression was also generated and compared to predicted interactions. Two global subnetworks were constructed for highly conserved interactions. These subnetworks showed clear clustering of proteins by function. Another subnetwork was created for disease response using a bait and prey strategy to capture interacting partners for proteins that respond to other organisms. Closer examination of this subnetwork revealed the connectivity between biotic and abiotic hormone stress pathways. We believe PiZeaM will provide a useful tool for the prediction of protein function and analysis of pathways for Z. mays researchers and is presented in this paper as a reference tool for the exploration of protein interactions in maize.

Variable timing of developmental progression in the stomatal pathway in Arabidopsis cotyledons.

• Stomatal production depends on the rates of precursor cell formation (e.g. the formation of meristemoids by asymmetric division), and of developmental progression (e.g. the division of guard mother cells). It is not known whether these rates follow steady-state kinetics or are variable. • The timing of development was scored in Arabidopsis cotyledons in fixed and living tissue using the dental resin impression method. • Cotyledons exhibited much less of a longitudinal gradient in stomatal formation than leaves. The timing of the appearance of stomatal and precursor cells during cotyledon development varied between individual plants. Precursor cell formation ceased much earlier in the adaxial than in the abaxial epidermis. Meristemoids are precursors that form guard mother cells. The ratio of these cell types varied greatly in different plants as well as in the same epidermis through time. There was also considerable variability in the duration of the meristemoid stage between individual cell lineages. • Precursor cell production follows non-steady-state kinetics. Early steps in the pathway are not necessarily synchronized, but later steps, such as the conversion of meristemoids to guard mother cells, sometimes appear to be coordinated.

Reproductive Toxicity and Life History Study of Silver Nanoparticle Effect, Uptake and Transport in Arabidopsis thaliana.

Concerns about nanotechnology have prompted studies on how the release of these engineered nanoparticles impact our environment. Herein, the impact of 20 nm silver nanoparticles (AgNPs) on the life history traits of Arabidopsis thaliana was studied in both above- and below-ground parts, at macroscopic and microscopic scales. Both gross phenotypes (in contrast to microscopic phenotypes) and routes of transport and accumulation were investigated from roots to shoots. Wild type Arabidopsis growing in soil, regularly irrigated with 75 µg/L of AgNPs, did not show any obvious morphological change. However, their vegetative development was prolonged by two to three days and their reproductive growth shortened by three to four days. In addition, the germination rates of offspring decreased drastically over three generations. These findings confirmed that AgNPs induce abiotic stress and cause reproductive toxicity in Arabidopsis. To trace transport of AgNPs, this study also included an Arabidopsis reporter line genetically transformed with a green fluorescent protein and grown in an optical transparent medium with 75 µg/L AgNPs. AgNPs followed three routes: (1) At seven days after planting (DAP) at S1.0 (stages defined by Boyes et al. 2001 [41]), AgNPs attached to the surface of primary roots and then entered their root tips; (2) At 14 DAP at S1.04, as primary roots grew longer, AgNPs gradually moved into roots and entered new lateral root primordia and root hairs; (3) At 17 DAP at S1.06 when the Arabidopsis root system had developed multiple lateral roots, AgNPs were present in vascular tissue and throughout the whole plant from root to shoot. In some cases, if cotyledons of the Arabidopsis seedlings were immersed in melted transparent medium, then AgNPs were taken up by and accumulated in stomatal guard cells. These findings in Arabidopsis are the first to document specific routes and rates of AgNP uptake in vivo and in situ.