John Danku

Variation in Molybdenum Content Across Broadly Distributed Populations of Arabidopsis thaliana Is Controlled by a Mitochondrial Molybdenum Transporter (MOT1)

Molybdenum (Mo) is an essential micronutrient for plants, serving as a cofactor for enzymes involved in nitrate assimilation, sulfite detoxification, abscisic acid biosynthesis, and purine degradation. Here we show that natural variation in shoot Mo content across 92 Arabidopsis thaliana accessions is controlled by variation in a mitochondrially localized transporter (Molybdenum Transporter 1 - MOT1) that belongs to the sulfate transporter superfamily. A deletion in the MOT1 promoter is strongly associated with low shoot Mo, occurring in seven of the accessions with the lowest shoot content of Mo. Consistent with the low Mo phenotype, MOT1 expression in low Mo accessions is reduced. Reciprocal grafting experiments demonstrate that the roots of Ler-0 are responsible for the low Mo accumulation in shoot, and GUS localization demonstrates that MOT1 is expressed strongly in the roots. MOT1 contains an N-terminal mitochondrial targeting sequence and expression of MOT1 tagged with GFP in protoplasts and transgenic plants, establishing the mitochondrial localization of this protein. Furthermore, expression of MOT1 specifically enhances Mo accumulation in yeast by 5-fold, consistent with MOT1 functioning as a molybdate transporter. This work provides the first molecular insight into the processes that regulate Mo accumulation in plants and shows that novel loci can be detected by association mapping.

Genome-Wide Association Studies Identify Heavy Metal ATPase3 as the Primary Determinant of Natural Variation in Leaf Cadmium in Arabidopsis thaliana

Understanding the mechanism of cadmium (Cd) accumulation in plants is important to help reduce its potential toxicity to both plants and humans through dietary and environmental exposure. Here, we report on a study to uncover the genetic basis underlying natural variation in Cd accumulation in a world-wide collection of 349 wild collected Arabidopsis thaliana accessions. We identified a 4-fold variation (0.5–2 µg Cd g−1 dry weight) in leaf Cd accumulation when these accessions were grown in a controlled common garden. By combining genome-wide association mapping, linkage mapping in an experimental F2 population, and transgenic complementation, we reveal that HMA3 is the sole major locus responsible for the variation in leaf Cd accumulation we observe in this diverse population of A. thaliana accessions. Analysis of the predicted amino acid sequence of HMA3 from 149 A. thaliana accessions reveals the existence of 10 major natural protein haplotypes. Association of these haplotypes with leaf Cd accumulation and genetics complementation experiments indicate that 5 of these haplotypes are active and 5 are inactive, and that elevated leaf Cd accumulation is associated with the reduced function of HMA3 caused by a nonsense mutation and polymorphisms that change two specific amino acids.

Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root.

Significance The endodermis acts as a “second skin” in plant roots by providing the cellular control necessary for the selective entry of water and mineral nutrients into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks diffusion across the endodermis in the cell wall. This junction is composed of a fine band of lignin, the polymer that gives wood its strength. Here, we characterize a dirigent protein (from Latin, dirigere: to guide or align) as playing a vital role in the patterning of lignin in the Casparian strip, identifying a new component of the molecular machinery that builds Casparian strips. The endodermis acts as a “second skin” in plant roots by providing the cellular control necessary for the selective entry of water and solutes into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks extracellular diffusion across the endodermis. This junction is composed of lignin that is polymerized by oxidative coupling of monolignols through the action of a NADPH oxidase and peroxidases. Casparian strip domain proteins (CASPs) correctly position this biosynthetic machinery by forming a protein scaffold in the plasma membrane at the site where the Casparian strip forms. Here, we show that the dirigent-domain containing protein, enhanced suberin1 (ESB1), is part of this machinery, playing an essential role in the correct formation of Casparian strips. ESB1 is localized to Casparian strips in a CASP-dependent manner, and in the absence of ESB1, disordered and defective Casparian strips are formed. In addition, loss of ESB1 disrupts the localization of the CASP1 protein at the casparian strip domain, suggesting a reciprocal requirement for both ESB1 and CASPs in forming the casparian strip domain

Analysis of the Root System Architecture of Arabidopsis Provides a Quantitative Readout of Crosstalk between Nutritional Signals

Root architectures of Arabidopsis thaliana were quantified in multiple combinations of N, P, K, S, and light to understand how plants integrate multiple nutritional signals. Combined with mutant phenotypes, transcriptional profiles, and shoot ion contents, the comprehensive data set facilitates the dissection of the nutrient signaling network and identifies roles for receptor-transporters. As plant roots forage the soil for food and water, they translate a multifactorial input of environmental stimuli into a multifactorial developmental output that manifests itself as root system architecture (RSA). Our current understanding of the underlying regulatory network is limited because root responses have traditionally been studied separately for individual nutrient deficiencies. In this study, we quantified 13 RSA parameters of Arabidopsis thaliana in 32 binary combinations of N, P, K, S, and light. Analysis of variance showed that each RSA parameter was determined by a typical pattern of environmental signals and their interactions. P caused the most important single-nutrient effects, while N-effects were strongly light dependent. Effects of K and S occurred mostly through nutrient interactions in paired or multiple combinations. Several RSA parameters were selected for further analysis through mutant phenotyping, which revealed combinations of transporters, receptors, and kinases acting as signaling modules in K–N interactions. Furthermore, nutrient response profiles of individual RSA features across NPK combinations could be assigned to transcriptionally coregulated clusters of nutrient-responsive genes in the roots and to ionome patterns in the shoots. The obtained data set provides a quantitative basis for understanding how plants integrate multiple nutritional stimuli into complex developmental programs.

Genome-wide Association Mapping Identifies a New Arsenate Reductase Enzyme Critical for Limiting Arsenic Accumulation in Plants

A genome-wide association study identifies the enzyme in plants that transforms arsenate into arsenite, allowing its extrusion into the soil and thereby controlling arsenic accumulation.

OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis

This work identifies a physiological substrate and a physiological function of the Arabidopsis oligopeptide transporter, OPT3, in iron (Fe) homeostasis, provides a mechanistic explanation of the role of OPT3 in systemic Fe signaling, and uncovers an aspect of crosstalk between Fe homeostasis and cadmium partitioning. Iron is essential for both plant growth and human health and nutrition. Knowledge of the signaling mechanisms that communicate iron demand from shoots to roots to regulate iron uptake as well as the transport systems mediating iron partitioning into edible plant tissues is critical for the development of crop biofortification strategies. Here, we report that OPT3, previously classified as an oligopeptide transporter, is a plasma membrane transporter capable of transporting transition ions in vitro. Studies in Arabidopsis thaliana show that OPT3 loads iron into the phloem, facilitates iron recirculation from the xylem to the phloem, and regulates both shoot-to-root iron signaling and iron redistribution from mature to developing tissues. We also uncovered an aspect of crosstalk between iron homeostasis and cadmium partitioning that is mediated by OPT3. Together, these discoveries provide promising avenues for targeted strategies directed at increasing iron while decreasing cadmium density in the edible portions of crops and improving agricultural productivity in iron deficient soils.

The MYB36 transcription factor orchestrates Casparian strip formation

Significance Casparian strips play a critical role in sealing endodermal cells in the root to block uncontrolled extracellular uptake of nutrients and water. Building Casparian strips requires the construction of extracellular lignin structures that encircle cells within the cell wall and that are anchored to the plasma membranes of adjacent cells to form tight seals between them. The transcription factor we have discovered, and the set of genes it regulates, now provides us with the detailed “parts list” necessary to build Casparian strips. This finding has clear implications for better understanding the nature of tight cellular junctions in biology and also has practical implications of agricultural, offering the potential for improved water and nutrient use efficiencies and enhanced resistance to abiotic stresses. The endodermis in roots acts as a selectivity filter for nutrient and water transport essential for growth and development. This selectivity is enabled by the formation of lignin-based Casparian strips. Casparian strip formation is initiated by the localization of the Casparian strip domain proteins (CASPs) in the plasma membrane, at the site where the Casparian strip will form. Localized CASPs recruit Peroxidase 64 (PER64), a Respiratory Burst Oxidase Homolog F, and Enhanced Suberin 1 (ESB1), a dirigent-like protein, to assemble the lignin polymerization machinery. However, the factors that control both expression of the genes encoding this biosynthetic machinery and its localization to the Casparian strip formation site remain unknown. Here, we identify the transcription factor, MYB36, essential for Casparian strip formation. MYB36 directly and positively regulates the expression of the Casparian strip genes CASP1, PER64, and ESB1. Casparian strips are absent in plants lacking a functional MYB36 and are replaced by ectopic lignin-like material in the corners of endodermal cells. The barrier function of Casparian strips in these plants is also disrupted. Significantly, ectopic expression of MYB36 in the cortex is sufficient to reprogram these cells to start expressing CASP1–GFP, correctly localize the CASP1–GFP protein to form a Casparian strip domain, and deposit a Casparian strip-like structure in the cell wall at this location. These results demonstrate that MYB36 is controlling expression of the machinery required to locally polymerize lignin in a fine band in the cell wall for the formation of the Casparian strip.

Variation in sulfur and selenium accumulation is controlled by naturally occurring isoforms of the key sulfur assimilation enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 across the arabidopsis species range

The ability of Arabidopsis to assimilate and accumulate sulfur varies widely across the species range and is controlled in part by diversity in a key reductase enzyme. Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored.

Single-Kernel Ionomic Profiles Are Highly Heritable Indicators of Genetic and Environmental Influences on Elemental Accumulation in Maize Grain (Zea mays)

The ionome, or elemental profile, of a maize kernel can be viewed in at least two distinct ways. First, the collection of elements within the kernel are food and feed for people and animals. Second, the ionome of the kernel represents a developmental end point that can summarize the life history of a plant, combining genetic programs and environmental interactions. We assert that single-kernel-based phenotyping of the ionome is an effective method of analysis, as it represents a reasonable compromise between precision, efficiency, and power. Here, we evaluate potential pitfalls of this sampling strategy using several field-grown maize sample sets. We demonstrate that there is enough genetically determined diversity in accumulation of many of the elements assayed to overcome potential artifacts. Further, we demonstrate that environmental signals are detectable through their influence on the kernel ionome. We conclude that using single kernels as the sampling unit is a valid approach for understanding genetic and environmental effects on the maize kernel ionome.

High-resolution genome-wide scan of genes, gene-networks and cellular systems impacting the yeast ionome

BackgroundTo balance the demand for uptake of essential elements with their potential toxicity living cells have complex regulatory mechanisms. Here, we describe a genome-wide screen to identify genes that impact the elemental composition (‘ionome’) of yeast Saccharomyces cerevisiae. Using inductively coupled plasma – mass spectrometry (ICP-MS) we quantify Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S and Zn in 11890 mutant strains, including 4940 haploid and 1127 diploid deletion strains, and 5798 over expression strains.ResultsWe identified 1065 strains with an altered ionome, including 584 haploid and 35 diploid deletion strains, and 446 over expression strains. Disruption of protein metabolism or trafficking has the highest likelihood of causing large ionomic changes, with gene dosage also being important. Gene over expression produced more extreme ionomic changes, but over expression and loss of function phenotypes are generally not related. Ionomic clustering revealed the existence of only a small number of possible ionomic profiles suggesting fitness tradeoffs that constrain the ionome. Clustering also identified important roles for the mitochondria, vacuole and ESCRT pathway in regulation of the ionome. Network analysis identified hub genes such as PMR1 in Mn homeostasis, novel members of ionomic networks such as SMF3 in vacuolar retrieval of Mn, and cross-talk between the mitochondria and the vacuole. All yeast ionomic data can be searched and downloaded at http://www.ionomicshub.org.ConclusionsHere, we demonstrate the power of high-throughput ICP-MS analysis to functionally dissect the ionome on a genome-wide scale. The information this reveals has the potential to benefit both human health and agriculture.