John D. Bussell

Phenotypic Plasticity of Adventitious Rooting in Arabidopsis Is Controlled by Complex Regulation of AUXIN RESPONSE FACTOR Transcripts and MicroRNA Abundance

The development of shoot-borne roots, or adventitious roots, is indispensable for mass propagation of elite genotypes. It is a complex genetic trait with a high phenotypic plasticity due to multiple endogenous and environmental regulatory factors. We demonstrate here that a subtle balance of activator and repressor AUXIN RESPONSE FACTOR (ARF) transcripts controls adventitious root initiation. Moreover, microRNA activity appears to be required for fine-tuning of this process. Thus, ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. The three ARFs display overlapping expression domains, interact genetically, and regulate each others expression at both transcriptional and posttranscriptional levels by modulating miR160 and miR167 availability. This complex regulatory network includes an unexpected feedback regulation of microRNA homeostasis by direct and nondirect target transcription factors. These results provide evidence of microRNA control of phenotypic variability and are a significant step forward in understanding the molecular mechanisms regulating adventitious rooting.

Auxin and Light Control of Adventitious Rooting in Arabidopsis Require ARGONAUTE1

Adventitious rooting is a quantitative genetic trait regulated by both environmental and endogenous factors. To better understand the physiological and molecular basis of adventitious rooting, we took advantage of two classes of Arabidopsis thaliana mutants altered in adventitious root formation: the superroot mutants, which spontaneously make adventitious roots, and the argonaute1 (ago1) mutants, which unlike superroot are barely able to form adventitious roots. The defect in adventitious rooting observed in ago1 correlated with light hypersensitivity and the deregulation of auxin homeostasis specifically in the apical part of the seedlings. In particular, a clear reduction in endogenous levels of free indoleacetic acid (IAA) and IAA conjugates was shown. This was correlated with a downregulation of the expression of several auxin-inducible GH3 genes in the hypocotyl of the ago1-3 mutant. We also found that the Auxin Response Factor17 (ARF17) gene, a potential repressor of auxin-inducible genes, was overexpressed in ago1-3 hypocotyls. The characterization of an ARF17-overexpressing line showed that it produced fewer adventitious roots than the wild type and retained a lower expression of GH3 genes. Thus, we suggest that ARF17 negatively regulates adventitious root formation in ago1 mutants by repressing GH3 genes and therefore perturbing auxin homeostasis in a light-dependent manner. These results suggest that ARF17 could be a major regulator of adventitious rooting in Arabidopsis.

Signaling from the endoplasmic reticulum activates brassinosteroid signaling and promotes acclimation to stress in Arabidopsis.

BR signaling may integrate stress responses and growth processes to optimize growth under challenging environmental conditions. Integrating Stress and Growth Brassinosteroids (BRs) are plant hormones that control various aspects of plant growth and development. BR signaling has also been implicated in plants’ ability to tolerate environmental stresses. Regulated intramembrane proteolysis (RIP) that occurs in the Golgi liberates membrane-bound transcription factors in response to various forms of cellular stress, such as endoplasmic reticulum (ER) stress, heat, and salt stress. Che et al. identified two transcription factors, bZIP17 and bZIP28, that were cleaved through endomembrane RIP and activated by heat and ER stress to mediate tolerance to stress. This RIP pathway enhanced BR signaling, suggesting a mechanism by which plants may optimize growth during challenging environmental conditions. The ability to acclimate to stresses enables plants to grow and develop under adverse environmental conditions. Regulated intramembrane proteolysis (RIP) triggered by endoplasmic reticulum (ER) stress mediates some forms of stress signaling. Brassinosteroids (BRs) have been implicated in plant adaptation to stress, but no mechanisms for activation have been discovered. Here, we reveal a connection between ER stress signaling and BR-mediated growth and stress acclimation. Arabidopsis transcription factors bZIP17 and bZIP28 were translocated from the ER through the Golgi, where they were proteolytically cleaved by site 2 protease and released to translocate into the nucleus. Stresses, including heat and inhibition of protein glycosylation, increased translocation of these two bZIPs to the nucleus. These nuclear-localized bZIPs not only activated ER chaperone genes but also activated BR signaling, which was required for stress acclimation and growth. Thus, these bZIPs link ER stress and BR signaling, which may be a mechanism by which plant growth and stress responses can be integrated.

Novel Proteins, Putative Membrane Transporters, and an Integrated Metabolic Network Are Revealed by Quantitative Proteomic Analysis of Arabidopsis Cell Culture Peroxisomes

Peroxisomes play key roles in energy metabolism, cell signaling, and plant development. A better understanding of these important functions will be achieved with a more complete definition of the peroxisome proteome. The isolation of peroxisomes and their separation from mitochondria and other major membrane systems have been significant challenges in the Arabidopsis (Arabidopsis thaliana) model system. In this study, we present new data on the Arabidopsis peroxisome proteome obtained using two new technical advances that have not previously been applied to studies of plant peroxisomes. First, we followed density gradient centrifugation with free-flow electrophoresis to improve the separation of peroxisomes from mitochondria. Second, we used quantitative proteomics to identify proteins enriched in the peroxisome fractions relative to mitochondrial fractions. We provide evidence for peroxisomal localization of 89 proteins, 36 of which have not previously been identified in other analyses of Arabidopsis peroxisomes. Chimeric green fluorescent protein constructs of 35 proteins have been used to confirm their localization in peroxisomes or to identify endoplasmic reticulum contaminants. The distribution of many of these peroxisomal proteins between soluble, membrane-associated, and integral membrane locations has also been determined. This core peroxisomal proteome from nonphotosynthetic cultured cells contains a proportion of proteins that cannot be predicted to be peroxisomal due to the lack of recognizable peroxisomal targeting sequence 1 (PTS1) or PTS2 signals. Proteins identified are likely to be components in peroxisome biogenesis, β-oxidation for fatty acid degradation and hormone biosynthesis, photorespiration, and metabolite transport. A considerable number of the proteins found in peroxisomes have no known function, and potential roles of these proteins in peroxisomal metabolism are discussed. This is aided by a metabolic network analysis that reveals a tight integration of functions and highlights specific metabolite nodes that most probably represent entry and exit metabolites that could require transport across the peroxisomal membrane.

The Arabidopsis Ortholog of Rice DWARF27 Acts Upstream of MAX1 in the Control of Plant Development by Strigolactones

Strigolactones (SLs) are carotenoid-derived plant hormones that regulate shoot branching, secondary growth, root development, and responses to soil phosphate. In Arabidopsis (Arabidopsis thaliana), SL biosynthesis requires the sequential action of two carotenoid cleavage dioxygenases, MORE AXILLARY GROWTH3 (MAX3) and MAX4, followed by a cytochrome P450, MAX1. In rice (Oryza sativa), the plastid-localized protein DWARF27 (OsD27) is also necessary for SL biosynthesis, but the equivalent gene in Arabidopsis has not been identified. Here, we use phylogenetic analysis of D27-like sequences from photosynthetic organisms to identify AtD27, the likely Arabidopsis ortholog of OsD27. Using reverse genetics, we show that AtD27 is required for the inhibition of secondary bud outgrowth and that exogenous application of the synthetic SL GR24 can rescue the increased branching phenotype of an Atd27 mutant. Furthermore, we use grafting to demonstrate that AtD27 operates on a nonmobile precursor upstream of MAX1 in the SL biosynthesis pathway. Consistent with the plastid localization of OsD27, we also show that AtD27 possesses a functional plastid transit peptide. We demonstrate that AtD27 transcripts are subject to both local feedback and auxin-dependent signals, albeit to a lesser extent than MAX3 and MAX4, suggesting that early steps in SL biosynthesis are coregulated at the transcriptional level. By identifying an additional component of the canonical SL biosynthesis pathway in Arabidopsis, we provide a new tool to investigate the regulation of shoot branching and other SL-dependent developmental processes.

A collection of INDEL markers for map-based cloning in seven Arabidopsis accessions.

The availability of a comprehensive set of resources including an entire annotated reference genome, sequenced alternative accessions, and a multitude of marker systems makes Arabidopsis thaliana an ideal platform for genetic mapping. PCR markers based on INsertions/DELetions (INDELs) are currently the most frequently used polymorphisms. For the most commonly used mapping combination, Columbia×Landsberg erecta (Col-0×Ler-0), the Cereon polymorphism database is a valuable resource for the generation of polymorphic markers. However, because the number of markers available in public databases for accessions other than Col-0 and Ler-0 is extremely low, mapping using other accessions is far from straightforward. This issue arose while cloning mutations in the Wassilewskija (Ws-4) background. In this work, approaches are described for marker generation in Ws-4 x Col-0. Complementary strategies were employed to generate 229 INDEL markers. Firstly, existing Col-0/Ler-0 Cereon predicted polymorphisms were mined for transferability to Ws-4. Secondly, Ws-0 ecotype Illumina sequence data were analyzed to identify INDELs that could be used for the development of PCR-based markers for Col-0 and Ws-4. Finally, shotgun sequencing allowed the identification of INDELs directly between Col-0 and Ws-4. The polymorphism of the 229 markers was assessed in seven widely used Arabidopsis accessions, and PCR markers that allow a clear distinction between the diverged Ws-0 and Ws-4 accessions are detailed. The utility of the markers was demonstrated by mapping more than 35 mutations in a Col-0×Ws-4 combination, an example of which is presented here. The potential contribution of next generation sequencing technologies to more traditional map-based cloning is discussed.

Identification of two Arabidopsis genes encoding a peroxisomal oxidoreductase-like protein and an acyl-CoA synthetase-like protein that are required for responses to pro-auxins

Indole-3-butyric acid (IBA) and 2,4-dichlorophenoxybutyric acid (2,4-DB) are metabolised by peroxisomal β-oxidation to active auxins that inhibit root growth. We screened Arabidopsis mutants for resistance to IBA and 2,4-DB and identified two new 2,4-DB resistant mutants. The mutant genes encode a putative oxidoreductase (SDRa) and a putative acyl-activating enzyme (AAE18). Both proteins are localised to peroxisomes. SDRa is coexpressed with core β-oxidation genes, but germination, seedling growth and the fatty acid profile of sdra seedlings are indistinguishable from wild type. The sdra mutant is also resistant to IBA, but aae18 is not. AAE18 is the first example of a gene required for response to 2,4-DB but not IBA. The closest relative of AAE18 is AAE17. AAE17 is predicted to be peroxisomal, but an aae17 aae18 double mutant responded similarly to aae18 for all assays. We propose that AAE18 is capable of activating 2,4-DB but IBA activating enzymes remain to be discovered. We present an updated model for peroxisomal pro-auxin metabolism in Arabidopsis that includes SDRa and AAE18.

An ecological genetic delineation of local seed‐source provenance for ecological restoration

An increasingly important practical application of the analysis of spatial genetic structure within plant species is to help define the extent of local provenance seed collection zones that minimize negative impacts in ecological restoration programs. Here, we derive seed sourcing guidelines from a novel range-wide assessment of spatial genetic structure of 24 populations of Banksia menziesii (Proteaceae), a widely distributed Western Australian tree of significance in local ecological restoration programs. An analysis of molecular variance (AMOVA) of 100 amplified fragment length polymorphism (AFLP) markers revealed significant genetic differentiation among populations (ΦPT = 0.18). Pairwise population genetic dissimilarity was correlated with geographic distance, but not environmental distance derived from 15 climate variables, suggesting overall neutrality of these markers with regard to these climate variables. Nevertheless, Bayesian outlier analysis identified four markers potentially under selection, although these were not correlated with the climate variables. We calculated a global R-statistic using analysis of similarities (ANOSIM) to test the statistical significance of population differentiation and to infer a threshold seed collection zone distance of ∼60 km (all markers) and 100 km (outlier markers) when genetic distance was regressed against geographic distance. Population pairs separated by >60 km were, on average, twice as likely to be significantly genetically differentiated than population pairs separated by <60 km, suggesting that habitat-matched sites within a 30-km radius around a restoration site genetically defines a local provenance seed collection zone for B. menziesii. Our approach is a novel probability-based practical solution for the delineation of a local seed collection zone to minimize negative genetic impacts in ecological restoration.

Peroxisomal ATP-Binding Cassette Transporter COMATOSE and the Multifunctional Protein ABNORMAL INFLORESCENCE MERISTEM Are Required for the Production of Benzoylated Metabolites in Arabidopsis Seeds

A peroxisomal substrate transporter and β-oxidation provide benzoic acid for the accumulation of benzoylated secondary metabolites during Arabidopsis seed development. Secondary metabolites derived from benzoic acid (BA) are of central importance in the interactions of plants with pests, pathogens, and symbionts and are potentially important in plant development. Peroxisomal β-oxidation has recently been shown to contribute to BA biosynthesis in plants, but not all of the enzymes involved have been defined. In this report, we demonstrate that the peroxisomal ATP-binding cassette transporter COMATOSE is required for the accumulation of benzoylated secondary metabolites in Arabidopsis (Arabidopsis thaliana) seeds, including benzoylated glucosinolates and substituted hydroxybenzoylcholines. The ABNORMAL INFLORESCENCE MERISTEM protein, one of two multifunctional proteins encoded by Arabidopsis, is essential for the accumulation of these compounds, and MULTIFUNCTIONAL PROTEIN2 contributes to the synthesis of substituted hydroxybenzoylcholines. Of the two major 3-ketoacyl coenzyme A thiolases, KAT2 plays the primary role in BA synthesis. Thus, BA biosynthesis in Arabidopsis employs the same core set of β-oxidation enzymes as in the synthesis of indole-3-acetic acid from indole-3-butyric acid.

Arabidopsis peroxisome proteomics

The analytical depth of investigation of the peroxisomal proteome of the model plant Arabidopsis thaliana has not yet reached that of other major cellular organelles such as chloroplasts or mitochondria. This is primarily due to the difficulties associated with isolating and obtaining purified samples of peroxisomes from Arabidopsis. So far only a handful of research groups have been successful in obtaining such fractions. To make things worse, enriched peroxisome fractions frequently suffer from significant organellar contamination, lowering confidence in localization assignment of the identified proteins. As with other cellular compartments, identification of peroxisomal proteins forms the basis for investigations of the dynamics of the peroxisomal proteome. It is therefore not surprising that, in terms of functional analyses by proteomic means, peroxisomes are lagging considerably behind chloroplasts or mitochondria. Alternative strategies are needed to overcome the obstacle of hard-to-obtain organellar fractions. This will help to close the knowledge gap between peroxisomes and other organelles and provide a full picture of the physiological pathways shared between organelles. In this review, we briefly summarize the status quo and discuss some of the methodological alternatives to classic organelle proteomic approaches.