Increased temperatures may safeguard the nutritional quality of crops under future elevated CO2 concentrations.

Iron (Fe) and zinc (Zn) deficiencies are a global human health problem that may worsen by the growth of crops at elevated atmospheric CO2 concentration (eCO2 ). However, climate change will also involve higher temperature, but it is unclear how the combined effect of eCO2 and higher temperature will affect the nutritional quality of food crops. To begin to address this question, we grew soybean (Glycine max) in a Temperature by Free-Air CO2 Enrichment (T-FACE) experiment in 2014 and 2015 under ambient (400 µmol mol-1 ) and elevated (600 µmol mol-1 ) CO2 concentrations, and under ambient and elevated temperatures (+2.7°C day and +3.4°C at night). In our study, eCO2 significantly decreased Fe concentration in soybean seeds in both seasons (-8.7 and -7.7%) and Zn concentration in one season (-8.9%), while higher temperature (at ambient CO2 concentration) had the opposite effect. The combination of eCO2 with elevated temperature generally restored seed Fe and Zn concentrations to levels obtained under ambient CO2 and temperature conditions, suggesting that the potential threat to human nutrition by increasing CO2 concentration may not be realized. In general, seed Fe concentration was negatively correlated with yield, suggesting inherent limitations to increasing seed Fe. In addition, we confirm our previous report that the concentration of seed storage products and several minerals varies with node position at which the seeds developed. Overall, these results demonstrate the complexity of predicting climate change effects on food and nutritional security when various environmental parameters change in an interactive manner.

Arabidopsis NPCC6/NaKR1 is a phloem mobile metal binding protein necessary for phloem function and root meristem maintenance.

SODIUM POTASSIUM ROOT DEFECTIVE1 (NaKR1; previously called NPCC6) encodes a soluble metal binding protein that is specifically expressed in companion cells of the phloem. The nakr1-1 mutant phenotype includes high Na(+), K(+), Rb(+), and starch accumulation in leaves, short roots, late flowering, and decreased long-distance transport of sucrose. Using traditional and DNA microarray-based deletion mapping, a 7-bp deletion was found in an exon of NaKR1 that introduced a premature stop codon. The mutant phenotypes were complemented by transformation with the native gene or NaKR1-GFP (green fluorescent protein) and NaKR1-ß-glucuronidase fusions driven by the native promoter. NAKR1-GFP was mobile in the phloem; it moved from companion cells into sieve elements and into a previously undiscovered symplasmic domain in the root meristem. Grafting experiments revealed that the high Na(+) accumulation was due mainly to loss of NaKR1 function in the leaves. This supports a role for the phloem in recirculating Na(+) to the roots to limit Na(+) accumulation in leaves. The onset of root phenotypes coincided with NaKR1 expression after germination. The nakr1-1 short root phenotype was due primarily to a decreased cell division rate in the root meristem, indicating a role in root meristem maintenance for NaKR1 expression in the phloem.

The ferroportin metal efflux proteins function in iron and cobalt homeostasis in Arabidopsis.

Relatively little is known about how metals such as iron are effluxed from cells, a necessary step for transport from the root to the shoot. Ferroportin (FPN) is the sole iron efflux transporter identified to date in animals, and there are two closely related orthologs in Arabidopsis thaliana, IRON REGULATED1 (IREG1/FPN1) and IREG2/FPN2. FPN1 localizes to the plasma membrane and is expressed in the stele, suggesting a role in vascular loading; FPN2 localizes to the vacuole and is expressed in the two outermost layers of the root in response to iron deficiency, suggesting a role in buffering metal influx. Consistent with these roles, fpn2 has a diminished iron deficiency response, whereas fpn1 fpn2 has an elevated iron deficiency response. Ferroportins also play a role in cobalt homeostasis; a survey of Arabidopsis accessions for ionomic phenotypes showed that truncation of FPN2 results in elevated shoot cobalt levels and leads to increased sensitivity to the metal. Conversely, loss of FPN1 abolishes shoot cobalt accumulation, even in the cobalt accumulating mutant frd3. Consequently, in the fpn1 fpn2 double mutant, cobalt cannot move to the shoot via FPN1 and is not sequestered in the root vacuoles via FPN2; instead, cobalt likely accumulates in the root cytoplasm causing fpn1 fpn2 to be even more sensitive to cobalt than fpn2 mutants.

The leaf ionome as a multivariable system to detect a plant's physiological status.

The contention that quantitative profiles of biomolecules contain information about the physiological state of the organism has motivated a variety of high-throughput molecular profiling experiments. However, unbiased discovery and validation of biomolecular signatures from these experiments remains a challenge. Here we show that the Arabidopsis thaliana (Arabidopsis) leaf ionome, or elemental composition, contains such signatures, and we establish statistical models that connect these multivariable signatures to defined physiological responses, such as iron (Fe) and phosphorus (P) homeostasis. Iron is essential for plant growth and development, but potentially toxic at elevated levels. Because of this, shoot Fe concentrations are tightly regulated and show little variation over a range of Fe concentrations in the environment, making them a poor probe of a plant's Fe status. By evaluating the shoot ionome in plants grown under different Fe nutritional conditions, we have established a multivariable ionomic signature for the Fe response status of Arabidopsis. This signature has been validated against known Fe-response proteins and allows the high-throughput detection of the Fe status of plants with a false negative/positive rate of 18%/16%. A "metascreen" of previously collected ionomic data from 880 Arabidopsis mutants and natural accessions for this Fe response signature successfully identified the known Fe mutants frd1 and frd3. A similar approach has also been taken to identify and use a shoot ionomic signature associated with P homeostasis. This study establishes that multivariable ionomic signatures of physiological states associated with mineral nutrient homeostasis do exist in Arabidopsis and are in principle robust enough to detect specific physiological responses to environmental or genetic perturbations.

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.

Leveraging non-targeted metabolite profiling via statistical genomics.

One of the challenges of systems biology is to integrate multiple sources of data in order to build a cohesive view of the system of study. Here we describe the mass spectrometry based profiling of maize kernels, a model system for genomic studies and a cornerstone of the agroeconomy. Using a network analysis, we can include 97.5% of the 8,710 features detected from 210 varieties into a single framework. More conservatively, 47.1% of compounds detected can be organized into a network with 48 distinct modules. Eigenvalues were calculated for each module and then used as inputs for genome-wide association studies. Nineteen modules returned significant results, illustrating the genetic control of biochemical networks within the maize kernel. Our approach leverages the correlations between the genome and metabolome to mutually enhance their annotation and thus enable biological interpretation. This method is applicable to any organism with sufficient bioinformatic resources.

Natural genetic variation in selected populations of Arabidopsis thaliana is associated with ionomic differences.

Controlling elemental composition is critical for plant growth and development as well as the nutrition of humans who utilize plants for food. Uncovering the genetic architecture underlying mineral ion homeostasis in plants is a critical first step towards understanding the biochemical networks that regulate a plant's elemental composition (ionome). Natural accessions of Arabidopsis thaliana provide a rich source of genetic diversity that leads to phenotypic differences. We analyzed the concentrations of 17 different elements in 12 A. thaliana accessions and three recombinant inbred line (RIL) populations grown in several different environments using high-throughput inductively coupled plasma- mass spectroscopy (ICP-MS). Significant differences were detected between the accessions for most elements and we identified over a hundred QTLs for elemental accumulation in the RIL populations. Altering the environment the plants were grown in had a strong effect on the correlations between different elements and the QTLs controlling elemental accumulation. All ionomic data presented is publicly available at www.ionomicshub.org.

Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana.

We used hybridization to the ATH1 gene expression array to interrogate genomic DNA diversity in 23 wild strains (accessions) of Arabidopsis thaliana (arabidopsis), in comparison with the reference strain Columbia (Col). At <1% false discovery rate, we detected 77,420 single-feature polymorphisms (SFPs) with distinct patterns of variation across the genome. Total and pair-wise diversity was higher near the centromeres and the heterochromatic knob region, but overall diversity was positively correlated with recombination rate (R(2) = 3.1%). The difference between total and pair-wise SFP diversity is a relative measure contrasting diversifying or frequency-dependent selection, similar to Tajima's D, and can be calibrated by the empirical genome-wide distribution. Each unique locus, centered on a gene, has a diversity and selection score that suggest a relative role in past evolutionary processes. Homologs of disease resistance (R) genes include members with especially high levels of diversity often showing frequency-dependent selection and occasionally evidence of a past selective sweep. Receptor-like and S-locus proteins also contained members with elevated levels of diversity and signatures of selection, whereas other gene families, bHLH, F-box, and RING finger proteins, showed more typical levels of diversity. SFPs identified with the gene expression array also provide an empirical hybridization polymorphism background for studies of gene expression polymorphism and are available through the genome browser http://signal.salk.edu/cgi-bin/AtSFP.

A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana.

The plasma membrane in plant cells is energized with an electrical potential and proton gradient generated through the action of H+ pumps belonging to the P-type ATPase superfamily. The Arabidopsis genome encodes 11 plasma membrane H+ pumps. Auto-inhibited H+-ATPase isoform 10 (AHA10) is expressed primarily in developing seeds. Here we show that four independent gene disruptions of AHA10 result in seed coats with a transparent testa (tt) phenotype (light-colored seeds). A quantitative analysis of extractable flavonoids in aha10 seeds revealed an approximately 100-fold reduction of proanthocyanidin (PA), one of the two major end-product pigments in the flavonoid biosynthetic pathway. In wild-type seed coat endothelial cells, PA accumulates in a large central vacuole. In aha10 mutants, the formation of this vacuole is impaired, as indicated by the predominance of multiple small vacuoles observed by fluorescence microscopy using a vacuole-specific dye, 5-(and -6)-carboxy 2',7'-dichlorofluorescein diacetate. A similar vacuolar defect was also observed for another tt mutant, tt12, a proton-coupled multidrug and toxic compound extrusion transporter potentially involved in loading provacuoles with a flavonoid intermediate required for PA production. The endothelial cells in aha10 mutants are otherwise healthy, as indicated by the lack of a significant decrease in (i) the accumulation of other flavonoid pathway end products, such as anthocyanins, and (ii) mRNA levels for two endothelium-specific transcripts (TT12 and BAN). Thus, the specific effect of aha10 on vacuolar and PA biogenesis provides genetic evidence to support an unexpected endomembrane function for a member of the plasma membrane H+-ATPase family.