The impact of Arabidopsis on human health: diversifying our portfolio

Studies of the model plant Arabidopsis thaliana may seem to have little impact on advances in medical research, yet a survey of the scientific literature shows that this is a misconception. Many discoveries with direct relevance to human health and disease have been elaborated using Arabidopsis, and several processes important to human biology are more easily studied in this versatile model plant.     

Chromosome landmarks as tools to study the genome of Arabidopsis thaliana

The model plant Arabidopsis thaliana has long been used for genetic, cellular and molecular studies. Whereas this plant was used as a model of genetics in the 1940's, the first cytogenetic observation of A. thaliana chromosomes was published in the beginning of the 20th century. Although Arabidopsis was not originally considered to be a good plant model for cytogenetics due to smallness of its genome, the number of published chromosome studies has expanded enormously in recent years. The advent of fluorescence in situ hybridization techniques on meiotic chromosomes together with indirect immuno-fluorescence localization of key chromosomal and nuclear proteins and wide accessibility of Arabidopsis mutants have resulted in a synergistic boost in Arabidopsis cytogenetics. In comparison to other plant species, the small genome with under-represented DNA repeats together with a small number of chromosomes makes this model plant easy to comprehend for a cytologist.     

Hormonal regulation of root growth: integrating local activities into global behaviour

To date, plant researchers have probed the control of root growth by studying the roles of individual regulatory components or cellular processes. However, recent studies in the Arabidopsis (Arabidopsis thaliana) root have shown that different hormones control organ growth by regulating specific growth processes (cell proliferation, differentiation or expansion) in distinct tissues. We discuss key issues raised by these new insights and hypothesise that novel tissue-to-tissue signals exist to coordinate organ growth. We conclude by describing how multiscale models can help probe the interplay between the different scales at which hormones and their regulatory networks operate in different cells and tissues. Such approaches promise to generate new insights into the mechanisms that control root growth.     

The plant Rad50-Mre11 protein complex

The Rad50-Mre11-Xrs2/Nbs1 protein complex plays critical roles in cellular processes involving DNA ends. This complex is implicated in DNA recombination and replication, meiosis, telomere maintenance and cellular DNA damage responses. The Rad50 and Mre11 proteins are essential for viability in animals, although not in yeast. We have prepared antibodies to the Rad50 protein of the model plant Arabidopsis thaliana which recognize a 175 kDa protein in wild-type Arabidopsis protein extracts. Furthermore, we report here demonstration of the existence of the Rad50-Mre11 complex by co-immunoprecipitation of the Rad50 and Mre11 proteins from the plant cell extracts.     

The dark side of the salad: Salmonella typhimurium overcomes the innate immune response of Arabidopsis thaliana and shows an endopathogenic lifestyle

Salmonella enterica serovar typhimurium contaminated vegetables and fruits are considerable sources of human infections. Bacteria present in raw plant-derived nutrients cause salmonellosis, the world wide most spread food poisoning. This facultative endopathogen enters and replicates in host cells and actively suppresses host immune responses. Although Salmonella survives on plants, the underlying bacterial infection mechanisms are only poorly understood. In this report we investigated the possibility to use Arabidopsis thaliana as a genetically tractable host system to study Salmonella-plant interactions. Using green fluorescent protein (GFP) marked bacteria, we show here that Salmonella can infect various Arabidopsis tissues and proliferate in intracellular cellular compartments. Salmonella infection of Arabidopsis cells can occur via intact shoot or root tissues resulting in wilting, chlorosis and eventually death of the infected organs. Arabidopsis reacts to Salmonella by inducing the activation of mitogen-activated protein kinase (MAPK) cascades and enhanced expression of pathogenesis related (PR) genes. The induction of defense responses fails in plants that are compromised in ethylene or jasmonic acid signaling or in the MKK3-MPK6 MAPK pathway. These findings demonstrate that Arabidopsis represents a true host system for Salmonella, offering unique possibilities to study the interaction of this human pathogen with plants at the molecular level for developing novel drug targets and addressing current safety issues in human nutrition.     

ATP-binding cassette B4, an auxin-efflux transporter, stably associates with the plasma membrane and shows distinctive intracellular trafficking from that of PIN-FORMED proteins

Intracellular trafficking of auxin transporters has been implicated in diverse developmental processes in plants. Although the dynamic trafficking pathways of PIN-FORMED auxin efflux proteins have been studied intensively, the trafficking of ATP-binding cassette protein subfamily B proteins (ABCBs; another group of auxin efflux carriers) still remains largely uncharacterized. In this study, we address the intracellular trafficking of ABCB4 in Arabidopsis (Arabidopsis thaliana) root epidermal cells. Pharmacological analysis showed that ABCB4 barely recycled between the plasma membrane and endosomes, although it slowly endocytosed via the lytic vacuolar pathway. Fluorescence recovery after photobleaching analysis revealed that ABCB4 is strongly retained in the plasma membrane, further supporting ABCB4's nonrecycling property. The endocytosis of ABCB4 was not dependent on the GNOM-LIKE1 function, and the sensitivity of ABCB4 to brefeldin A required guanine nucleotide exchange factors for adenosyl ribosylation factor other than GNOM. These characteristics of intracellular trafficking of ABCB4 are well contrasted with those of PIN-FORMED proteins, suggesting that ABCB4 may be a basic and constitutive auxin efflux transporter for cellular auxin homeostasis.     

A kaleidoscopic view of the Arabidopsis core cell cycle interactome

Although protein-protein interaction (PPI) networks have been shown to offer a systems-wide view of cellular processes, only a few plant PPI maps are available. Recently, the core cell cycle of Arabidopsis thaliana has been analyzed by three independent PPI technologies, including yeast two-hybrid systems, bimolecular fluorescence complementation and tandem affinity purification. Here, we merge the three interactomes with literature-curated and computationally predicted interactions, paving the way for a comprehensive picture of the plant core cell cycle machinery. Platform-specific interactions unveil the strengths and weaknesses of each detection method and give insights into the nature of the interactions among cell cycle proteins. Moreover, comparison of the obtained data reveals that a complete interactome can only be obtained when multiple techniques are applied in parallel.     

CLENCH: a program for calculating Cluster ENriCHment using the Gene Ontology

SUMMARY: Analysis of microarray data most often produces lists of genes with similar expression patterns, which are then subdivided into functional categories for biological interpretation. Such functional categorization is most commonly accomplished using Gene Ontology (GO) categories. Although there are several programs that identify and analyze functional categories for human, mouse and yeast genes, none of them accept Arabidopsis thaliana data. In order to address this need for A.thaliana community, we have developed a program that retrieves GO annotations for A.thaliana genes and performs functional category analysis for lists of genes selected by the user. AVAILABILITY: http://www.personal.psu.edu/nhs109/Clench     

Comprehensive analysis of CLE polypeptide signaling gene expression and overexpression activity in Arabidopsis

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.     

Putative spermine synthases from Thalassiosira pseudonana and Arabidopsis thaliana synthesize thermospermine rather than spermine

Polyamines are involved in many fundamental cellular processes. Common polyamines are putrescine, spermidine and spermine. Spermine is synthesized by transfer of an aminopropyl residue derived from decarboxylated S-adenosylmethionine to spermidine. Thermospermine is an isomer of spermine and assumed to be synthesized by an analogous mechanism. However, none of the recently described spermine synthases was investigated for their possible activity as thermospermine synthases. In this work, putative spermine synthases from the diatom Thalassiosira pseudonana and from Arabidopsis thaliana could be identified as thermospermine synthases. These findings may explain the previous result that two putative spermine synthase genes in Arabidopsis produce completely different phenotypes in knock-out experiments. Likely, part of putative spermine synthases identifiable by sequence comparisons represents in fact thermospermine synthases.     

Short-chain dehydrogenase/reductase (SDR) relationships: a large family with eight clusters common to human, animal, and plant genomes

The progress in genome characterizations has opened new routes for studying enzyme families. The availability of the human genome enabled us to delineate the large family of short-chain dehydrogenase/reductase (SDR) members. Although the human genome releases are not yet final, we have already found 63 members. We have also compared these SDR forms with those of three model organisms: Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana. We detect eight SDR ortholog clusters in a cross-genome comparison. Four of these clusters represent extended SDR forms, a subgroup found in all life forms. The other four are classical SDRs with activities involved in cellular differentiation and signalling. We also find 18 SDR genes that are present only in the human genome of the four genomes studied, reflecting enzyme forms specific to mammals. Close to half of these gene products represent steroid dehydrogenases, emphasizing the regulatory importance of these enzymes.     

Flavonoids:New Roles for Old Molecules

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions including defense, UV protection, auxin transport inhibition, allelopathy, and flower coloring. Interestingly, these compounds also have considerable biological activity in plant, animal and bacterial systems – such broad activity is accomplished by few compounds. Yet, for all the research over the last three decades, many of the cellular targets of these secondary metabolites are unknown. The many mutants available in model plant species such as Arabidopsis thaliana and Medicago truncatula are enabling the intricacies of the physiology of these compounds to be deduced. In the present review, we cover recent advances in flavonoid research, discuss deficiencies in our understanding of the physiological processes, and suggest approaches to identify the cellular targets of flavonoids.     

Proteomic identification of S-nitrosylated proteins in Arabidopsis

Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S-nitrosoglutathione. Furthermore, Arabidopsis plants were treated with gaseous NO to analyze whether S-nitrosylation can occur in the specific redox environment of a plant cell in vivo. S-Nitrosylated proteins were detected by a biotin switch method, converting S-nitrosylated Cys to biotinylated Cys. Biotin-labeled proteins were purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic proteins. Strikingly, many of these proteins have been identified previously as targets of S-nitrosylation in animals. At the enzymatic level, a case study demonstrated NO-dependent reversible inhibition of plant glyceraldehyde-3-phosphate dehydrogenase, suggesting that this enzyme could be affected by S-nitrosylation. The results of this work are the starting point for further investigation to get insight into signaling pathways and other cellular processes regulated by protein S-nitrosylation in plants.     

Genome-wide comparison of nucleotide-binding site-leucine-rich repeat-encoding genes in Arabidopsis

Plants, like animals, use several lines of defense against pathogen attack. Prominent among genes that confer disease resistance are those encoding nucleotide-binding site-leucine-rich repeat (NB-LRR) proteins. Likely due to selection pressures caused by pathogens, NB-LRR genes are the most variable gene family in plants, but there appear to be species-specific limits to the number of NB-LRR genes in a genome. Allelic diversity within an individual is also increased by obligatory outcrossing, which leads to genome-wide heterozygosity. In this study, we compared the NB-LRR gene complement of the selfer Arabidopsis thaliana and its outcrossing close relative Arabidopsis lyrata. We then complemented and contrasted the interspecific patterns with studies of NB-LRR diversity within A. thaliana. Three important insights are as follows: (1) that both species have similar numbers of NB-LRR genes; (2) that loci with single NB-LRR genes are less variable than tandem arrays; and (3) that presence-absence polymorphisms within A. thaliana are not strongly correlated with the presence or absence of orthologs in A. lyrata. Although A. thaliana individuals are mostly homozygous and thus potentially less likely to suffer from aberrant interaction of NB-LRR proteins with newly introduced alleles, the number of NB-LRR genes is similar to that in A. lyrata. In intraspecific and interspecific comparisons, NB-LRR genes are also more variable than receptor-like protein genes. Finally, in contrast to Drosophila, there is a clearly positive relationship between interspecific divergence and intraspecific polymorphisms.     

AtKP1, a kinesin-like protein, mainly localizes to mitochondria in Arabidopsis thaliana

Kinesins and kinesin-like proteins （KLPs） constitute a large family of microtubule-based motors that play important roles in many fundamental cellular and developmental processes. To date, a number of kinesins or KLPs have been identified in plants including Arabidopsis thaliana. Here, a polyclonal antibody against AtKP1 （kinesin-like protein 1 in A. thaliana） was raised by injection the expressed AtKP1 specific C-terminal polypeptides in rabbits, and immunoblot analysis was conducted with the affinity-purified anti-AtKP1 antibody. The results indicated that this antibody recognized the AtKP1 fusion proteins expressed in E. coli and proteins of -125 kDa in the soluble fractions of Arabidopsis extracts. The molecular weight was consistent with the calculated molecular weight based on deduced amino acids sequence of AtKP1. To acquire the subcellular localization of the protein, AtKP1 in Arabidopsis root cells was observed by indirect immunofluorescence microscopy. AtKP1 was localized to particle-like organelles in interphase or dividing cells, but not to mitotic microtubule arrays. Relatively more AtKP1 was found in isolated mitochondria fraction on immunoblot of the subcellular fractions. The AtKP1 protein could not be released following a 0.6 M KI washing, indicating that AtKP1 is tightly bind to mitochondria and might function associated with this kind of organelles.     

Analysis of protein interactions within the cytokinin-signaling pathway of Arabidopsis thaliana

The signal of the plant hormone cytokinin is perceived by membrane-located sensor histidine kinases and transduced by other members of the plant two-component system. In Arabidopsis thaliana, 28 two-component system proteins (phosphotransmitters and response regulators) act downstream of three receptors, transmitting the signal from the membrane to the nucleus and modulating the cellular response. Although the principal signaling mechanism has been elucidated, redundancy in the system has made it difficult to understand which of the many components interact to control the downstream biological processes. Here, we present a large-scale interaction study comprising most members of the Arabidopsis cytokinin signaling pathway. Using the yeast two-hybrid system, we detected 42 new interactions, of which more than 90% were confirmed by in vitro coaffinity purification. There are distinct patterns of interaction between protein families, but only a few interactions between proteins of the same family. An interaction map of this signaling pathway shows the Arabidopsis histidine phosphotransfer proteins as hubs, which interact with members from all other protein families, mostly in a redundant fashion. Domain-mapping experiments revealed the interaction domains of the proteins of this pathway. Analyses of Arabidopsis histidine phosphotransfer protein 5 mutant proteins showed that the presence of the canonical phospho-accepting histidine residue is not required for the interactions. Interaction of A-type response regulators with Arabidopsis histidine phosphotransfer proteins but not with B-type response regulators suggests that their known activity in feedback regulation may be realized by interfering at the level of Arabidopsis histidine phosphotransfer protein-mediated signaling. This study contributes to our understanding of the protein interactions of the cytokinin-signaling system and provides a framework for further functional studies in planta.