Daniel R. Ruzicka

A genome triplication associated with early diversification of the core eudicots.

BackgroundAlthough it is agreed that a major polyploidy event, gamma, occurred within the eudicots, the phylogenetic placement of the event remains unclear.ResultsTo determine when this polyploidization occurred relative to speciation events in angiosperm history, we employed a phylogenomic approach to investigate the timing of gene set duplications located on syntenic gamma blocks. We populated 769 putative gene families with large sets of homologs obtained from public transcriptomes of basal angiosperms, magnoliids, asterids, and more than 91.8 gigabases of new next-generation transcriptome sequences of non-grass monocots and basal eudicots. The overwhelming majority (95%) of well-resolved gamma duplications was placed before the separation of rosids and asterids and after the split of monocots and eudicots, providing strong evidence that the gamma polyploidy event occurred early in eudicot evolution. Further, the majority of gene duplications was placed after the divergence of the Ranunculales and core eudicots, indicating that the gamma appears to be restricted to core eudicots. Molecular dating estimates indicate that the duplication events were intensely concentrated around 117 million years ago.ConclusionsThe rapid radiation of core eudicot lineages that gave rise to nearly 75% of angiosperm species appears to have occurred coincidentally or shortly following the gamma triplication event. Reconciliation of gene trees with a species phylogeny can elucidate the timing of major events in genome evolution, even when genome sequences are only available for a subset of species represented in the gene trees. Comprehensive transcriptome datasets are valuable complements to genome sequences for high-resolution phylogenomic analysis.

Arabidopsis Actin-Depolymerizing Factor AtADF4 Mediates Defense Signal Transduction Triggered by the Pseudomonas syringae Effector AvrPphB

The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes with limited, indirect evidence. To date, there are no reports linking actin with resistance against phytopathogenic bacteria. The dynamic behavior of actin filaments is regulated by a diverse array of actin-binding proteins, among which is the Actin-Depolymerizing Factor (ADF) family of proteins. Here, we demonstrate that actin dynamics play a role in the activation of gene-for-gene resistance in Arabidopsis (Arabidopsis thaliana) following inoculation with the phytopathogenic bacterium Pseudomonas syringae pv tomato. Using a reverse genetics approach, we explored the roles of Arabidopsis ADFs in plant defenses. AtADF4 was identified as being specifically required for resistance triggered by the effector AvrPphB but not AvrRpt2 or AvrB. Recombinant AtADF4 bound to monomeric actin (G-actin) with a marked preference for the ADP-loaded form and inhibited the rate of nucleotide exchange on G-actin, indicating that AtADF4 is a bona fide actin-depolymerizing factor. Exogenous application of the actin-disrupting agent cytochalasin D partially rescued the Atadf4 mutant in the AvrPphB-mediated hypersensitive response, demonstrating that AtADF4 mediates defense signaling through modification of the actin cytoskeleton. Unlike the mechanism by which the actin cytoskeleton confers resistance against fungi and oomycetes, AtADF4 is not involved in resistance against pathogen entry. Collectively, this study identifies AtADF4 as a novel component of the plant defense signaling pathway and provides strong evidence for actin dynamics as a primary component that orchestrates plant defenses against P. syringae.

The Arabidopsis AP2/ERF Transcription Factor RAP2.11 Modulates Plant Response to Low-Potassium Conditions

Plants respond to low-nutrient conditions through metabolic and morphology changes that increase their ability to survive and grow. The transcription factor RAP2.11 was identified as a component in the response to low potassium through regulation of the high-affinity K(+) uptake transporter AtHAK5 and other components of the low-potassium signal transduction pathway. RAP2.11 was identified through the activation tagging of Arabidopsis lines that contained a luciferase marker driven by the AtHAK5 promoter that is normally only induced by low potassium. This factor bound to a GCC-box of the AtHAK5 promoter in vitro and in vivo. Transcript profiling revealed that a large number of genes were up-regulated in roots by RAP2.11 overexpression. Many regulated genes were identified to be in functional categories that are important in low-K(+) signaling. These categories included ethylene signaling, reactive oxygen species production, and calcium signaling. Promoter regions of the up-regulated genes were enriched in the GCCGGC motif also contained in the AtHAK5 promoter. These results suggest that RAP2.11 regulates AtHAK5 expression under low-K(+) conditions and also contributes to a coordinated response to low-potassium conditions through the regulation of other genes in the low-K(+) signaling cascade.

Class-specific interaction of profilin and ADF isovariants with actin in the regulation of plant development.

Two ancient and highly divergent actin-based cytoskeletal systems have evolved in angiosperms. Plant genomes encode complex actin and actin binding protein (ABP) gene families, most of which are phylogenetically grouped into gene classes with distinct vegetative or constitutive and reproductive expression patterns. In Arabidopsis thaliana, ectopic expression of high levels of a reproductive class actin, ACT1, in vegetative tissues causes severe dwarfing of plants with aberrant organization of most plant organs and cell types due to a severely altered actin cytoskeletal architecture. Overexpression of the vegetative class actin ACT2 to similar levels, however, produces insignificant phenotypic changes. We proposed that the misexpression of the pollen-specific ACT1 in vegetative cell types affects the dynamics of actin due to its inappropriate interaction with endogenous vegetative ABPs. To examine the functionally distinct interactions among the major classes of actins and ABPs, we ectopically coexpressed reproductive profilin (PRF4) or actin-depolymerizing factor (ADF) isovariants (e.g., ADF7) with ACT1. Our results demonstrated that the coexpression of these reproductive, but not vegetative, ABP isovariants suppressed the ectopic ACT1 expression phenotypes and restored wild-type stature and normal actin cytoskeletal architecture to the double transgenic plants. Thus, the actins and ABPs appear to have evolved class-specific, protein–protein interactions that are essential to the normal regulation of plant growth and development.

Challenges of modifying root traits in crops for agriculture.

Roots play an essential role in the acquisition of water and minerals from soils. Measuring crop root architecture and assaying for changes in function can be challenging, but examples have emerged showing that modifications to roots result in higher yield and increased stress tolerance. In this review, we focus mainly on the molecular genetic advances that have been made in altering root system architecture and function in crop plants, as well as phenotyping methods. The future for the modification of crop plant roots looks promising based on recent advances, but there are also important challenges ahead.

Transcriptomic and metabolic responses of mycorrhizal roots to nitrogen patches under field conditions

BackgroundArbuscular mycorrhizal (AM) fungi contribute to plant nutrient uptake in systems managed with reduced fertilizer and pesticide inputs such as organic agriculture by extending the effective size of the rhizosphere and delivering minerals to the root. Connecting the molecular study of the AM symbiosis with agriculturally- and ecologically-relevant field environments remains a challenge and is a largely unexplored research topic.MethodsThis study utilized a cross-disciplinary approach to examine the transcriptional, metabolic, and physiological responses of tomato (Solanum lycopersicum) AM roots to a localized patch of nitrogen (N). A wild-type mycorrhizal tomato and a closely-related non-mycorrhizal mutant were grown at an organic farm in soil that contained an active AM extraradical hyphal network and soil microbe community.ResultsThe majority of genes regulated by upon enrichment of nitrogen were similarly expressed in mycorrhizal and non-mycorrhizal roots, suggesting that the primary response to an enriched N patch is mediated by mycorrhiza-independent root processes. However where inorganic N concentrations in the soil were low, differential regulation of key tomato N transport and assimilation genes indicate a transcriptome shift towards mycorrhiza-mediated N uptake over direct root supplied N. Furthermore, two novel mycorrhizal-specific tomato ammonium transporters were also found to be regulated under low N conditions. A conceptual model is presented integrating the transcriptome response to low N and highlighting the mycorrhizal-specific ammonium transporters.ConclusionsThese results enhance our understanding of the role of the AM symbiosis in sensing and response to an enriched N patch, and demonstrate that transcriptome analyses of complex plant-microbe-soil interactions provide a global snapshot of biological processes relevant to soil processes in organic agriculture.

ACTIN DEPOLYMERIZING FACTOR9 controls development and gene expression in Arabidopsis

Actin depolymerizing factors (ADF/cofilin) modulate the rate of actin filament turnover, networking cellular signals into cytoskeletal-dependent developmental pathways. Plant and animal genomes encode families of diverse ancient ADF isovariants. One weakly but ubiquitously expressed member of the Arabidopsis ADF gene family, ADF9, is moderately expressed in the shoot apical meristem (SAM). Mutant alleles adf9-1 and adf9-2 showed a 95% and 50% reduction in transcript levels, respectively. Compared to wild-type, mutant seedlings and plants were significantly smaller and adult mutant plants had decreased numbers of lateral branches and a reduced ability to form callus. The mutants flowered very early during long-day light cycles, but not during short days. adf9-1showed a several-fold lower expression of FLOWERING LOCUS C (FLC), a master repressor of the transition to flowering, and increased expression of CONSTANS, an activator of flowering. Transgenic ADF9 expression complemented both developmental and gene expression phenotypes. FLC chromatin from adf9-1 plants contained reduced levels of histone H3 lysine 4 trimethylation and lysine 9 and 14 acetylation, as well as increased nucleosome occupancy consistent with a less active chromatin state. We propose that ADF9 networks both cytoplasmic and nuclear processes within the SAM to control multicellular development.

Inside Arbuscular Mycorrhizal Roots - Molecular Probes to Understand the Symbiosis

Associations between arbuscular mycorrhizal (AM) fungi and plants are an ancient and widespread plant microbe symbioses. Most land plants can associate with this specialized group of soil fungi (in the Glomeromycota), which enhance plant nutrient uptake in return for C derived from plant photosynthesis. Elucidating the mechanisms involved in the symbiosis between obligate symbionts such as AM fungi and plant roots is challenging because AM fungal transcripts in roots are in low abundance and reference genomes for the fungi have not been available. A deep sequencing metatranscriptomics approach was applied to a wild‐type tomato and a tomato mutant (Solanum lycopersicum L. cultivar RioGrande 76R) incapable of supporting a functional AM symbiosis, revealing novel AM fungal and microbial transcripts expressed in colonized roots. We confirm transcripts known to be mycorrhiza associated and report the discovery of more than 500 AM fungal and novel plant transcripts associated with mycorrhizal tomato roots including putative Zn, Fe, aquaporin, and carbohydrate transporters as well as mycorrhizal‐associated alternative gene splicing. This analysis provides a fundamental step toward identifying the molecular mechanisms of mineral and carbohydrate exchange during the symbiosis. The utility of this metatranscriptomic approach to explore an obligate biotrophic interaction is illustrated, especially as it relates to agriculturally relevant biological processes.