Iron (Fe) and zinc (Zn) are essential micronutrients required for proper development in both humans and plants. Rice (Oryza sativa L.) grains are the staple food for nearly half of the world's population, but a poor source of metals such as Fe and Zn. Populations that rely on milled cereals are especially prone to Fe and Zn deficiencies, the most prevalent nutritional deficiencies in humans. Biofortification is a cost-effective solution for improvement of the nutritional quality of crops. However, a better understanding of the mechanisms underlying grain accumulation of mineral nutrients is required before this approach can achieve its full potential. Characterization of gene function is more time-consuming in crops than in model species such as Arabidopsis thaliana. Aiming to more quickly characterize rice genes related to metal homeostasis, we applied the concept of high throughput elemental profiling (ionomics) to Arabidopsis lines heterologously expressing rice cDNAs driven by the 35S promoter, named FOX (Full Length Over-eXpressor) lines. We screened lines expressing candidate genes that could be used in the development of biofortified grain. Among the most promising candidates, we identified two lines ovexpressing the metal cation transporter OsZIP7. OsZIP7 expression in Arabidopsis resulted in a 25% increase in shoot Zn concentrations compared to non-transformed plants. We further characterized OsZIP7 and showed that it is localized to the plasma membrane and is able to complement Zn transport defective (but not Fe defective) yeast mutants. Interestingly, we showed that OsZIP7 does not transport Cd, which is commonly transported by ZIP proteins. Importantly, OsZIP7-expressing lines have increased Zn concentrations in their seeds. Our results indicate that OsZIP7 is a good candidate for developing Zn biofortified rice. Moreover, we showed the use of heterologous expression of genes from crops in A. thaliana as a fast method for characterization of crop genes related to the ionome and potentially useful in biofortification strategies.

The formation of Casparian strips and suberin lamellae at the endodermis limits the free diffusion of nutrients and harmful substances via the apoplastic space between the soil solution and the stele in roots [1-3]. Casparian strips are ring-like lignin polymers deposited in the middle of anticlinal cell walls between endodermal cells and fill the gap between them [4-6]. Suberin lamellae are glycerolipid polymers covering the endodermal cells and likely function as a barrier to limit transmembrane movement of apoplastic solutes into the endodermal cells [7, 8]. However, the current knowledge on the formation of these two distinct endodermal barriers and their regulatory role in nutrient transport is still limited. Here, we identify an uncharacterized gene, LOTR1, essential for Casparian strip formation in Arabidopsis thaliana. The lotr1 mutants display altered localization of CASP1, an essential protein for Casparian strip formation [9], disrupted Casparian strips, ectopic suberization of endodermal cells, and low accumulation of shoot calcium (Ca). Degradation by expression of a suberin-degrading enzyme in the mutants revealed that the ectopic suberization at the endodermal cells limits Ca transport through the transmembrane pathway, thereby causing reduced Ca delivery to the shoot. Moreover, analysis of the mutants showed that suberin lamellae function as an apoplastic diffusion barrier to the stele at sites of lateral root emergence where Casparian strips are disrupted. Our findings suggest that the transmembrane pathway through unsuberized endodermal cells, rather than the sites of lateral root emergence, mediates the transport of apoplastic substances such as Ca into the xylem.

Zinc (Zn) is an essential nutrient for plants, with a crucial role as a cofactor for many enzymes. Approximately one-third of the global arable land area is Zn deficient, leading to reduced crop yield and quality. To improve crop tolerance to Zn deficiency, it is important to understand the mechanisms plants have adopted to tolerate suboptimal Zn supply. In this study, physiological and molecular aspects of traits related to Zn deficiency tolerance were examined in a panel of 19 Arabidopsis thaliana accessions. Accessions showed a larger variation for shoot biomass than for Zn concentration, indicating that they have different requirements for their minimal Zn concentration required for growth. Accessions with a higher tolerance to Zn deficiency showed an increased expression of the Zn deficiency-responsive genes ZIP4 and IRT3 in comparison with Zn deficiency-sensitive accessions. Changes in the shoot ionome, as a result of the Zn treatment of the plants, were used to build a multinomial logistic regression model able to distinguish plants regarding their Zn nutritional status. This set of biomarkers, reflecting the A. thaliana response to Zn deficiency and Zn deficiency tolerance, can be useful for future studies aiming to improve the performance and Zn status of crop plants grown under suboptimal Zn concentrations.

As the world's population increases, demands on staple crops like rice (Oryza sativa L.) will also increase, requiring additional fresh water supplies for irrigation of rice fields. Safe alternate wetting and drying (AWD) is a water management technique that is being adopted across a number of countries to reduce the water input for rice cultivation. The impact of AWD on plant growth, yield and grain quality is not well understood. A field trial of AWD was conducted at Mymensingh, Bangladesh over two boro (dry) seasons using eight field plots, four under AWD and four continuously flooded (CF). This manuscript describes the results of check cultivar BRRI dhan28 which was replicated in 35-40 rows per plot giving a total of 140-160 replicates per treatment. A study on the soil solution concentration of many elements indicated that manganese, iron, zinc, and arsenic were different under AWD conditions compared to CF on a number of sampling time points, but did not show a pattern related to the AWD treatment. A survey of soil strength using a penetrometer detected a small, but statistically significant, hardening of the surface soil of the AWD plots. At harvest the shoot and grain mass was significantly greater for the plants grown under AWD (9.0-9.4% and 12.0-15.4%, respectively) with the plants grown under AWD having a greater number of productive tillers. Physiological examination in the first year showed that although AWD decreased (similar to 21%) leaf elongation rate (LER) of recently transplanted seedlings during the first drying cycle, subsequent drying cycles did not affect LER, while tillering was slightly increased by AWD and there was evidence of higher leaf abscisic acid (ABA) in AWD plants. In the second year analysis of six phytohormones revealed that AWD increased plant foliar isopentenyladenine (iP) concentrations by 37% while leaf trans-zeatin concentrations decreased (36%) compared to CF plants. The elemental composition of the shoots and grains was also examined. In both years AWD decreased grain concentration of sulphur (by 4% and 15%), calcium (by 6% and 9%), iron (by 11% and 16%), and arsenic (by 14% and 26%), while it increased the grain concentration of manganese (by 19% and 28%), copper (by 81% and 37%), and cadmium (by 28% and 67%). These results indicate that plants grown under safe AWD conditions at this site have an increased grain mass compared to plants grown under CF, and this may be partly due to a high number of productive tillers. AWD decreases the concentration of arsenic in the grains in this site, but it elevates the concentration of cadmium. (C) 2017 Elsevier B.V. All rights reserved.

As the global population grows, demand on food production will also rise. For rice, one limiting factor effecting production could be availability of fresh water, hence adoption of techniques that decrease water usage while maintaining or increasing crop yield are needed. Alternative wetting and drying (AWD) is one of these techniques. AWD is a method by which the level of water within a rice field cycles between being flooded and nonflooded during the growth period of the rice crop. The degree to which AWD affects cultivars differently has not been adequately addressed to date. In this study, 22 rice cultivars, mostly landraces of the aus subpopulation, plus some popular improved indica cultivars from Bangladesh, were tested for their response to AWD across three different field sites in Bangladesh. Grain and shoot elemental concentrations were determined at harvest. Overall, AWD slightly increased grain mass and harvest index compared to plants grown under continually flooded (CF) conditions. Plants grown under AWD had decreased concentrations of nitrogen in their straw compared to plants grown under CF. The concentration of elements in the grain were also affected when plants were grown under AWD compared to CF: Nickel, copper, cadmium and iron increased, but sodium, potassium, calcium, cobalt, phosphorus, molybdenum and arsenic decreased in the grains of plants grown under AWD. However, there was some variation in these patterns across different sites. Analysis of variance revealed no significant cultivar × treatment interaction, or site × cultivar × treatment interaction, for any of the plant mass traits. Of the elements analyzed, only grain cadmium concentrations were significantly affected by treatment × cultivar interactions. These data suggest that there is no genetic adaptation amongst the cultivars screened for response to AWD, except for grain cadmium concentration and imply that breeding specifically for AWD is not needed.

Rice is a major source of calories and mineral nutrients for over half the world's human population. However, little is known in rice about the genetic basis of variation in accumulation of copper (Cu), an essential but potentially toxic nutrient. Here we identify OsHMA4 as the likely causal gene of a quantitative trait locus controlling Cu accumulation in rice grain. We provide evidence that OsHMA4 functions to sequester Cu into root vacuoles, limiting Cu accumulation in the grain. The difference in grain Cu accumulation is most likely attributed to a single amino acid substitution that leads to different OsHMA4 transport activity. The allele associated with low grain Cu was found in 67 of the 1,367 rice accessions investigated. Identification of natural allelic variation in OsHMA4 may facilitate the development of rice varieties with grain Cu concentrations tuned to both the concentration of Cu in the soil and dietary needs.

Cadmium (Cd) is a heavy metal toxic to humans and its occurrence in soils represents a significant environmental problem. Poplar trees may provide one possible option to help remove Cd contamination from soil. However, before this is practicable, the ability of poplar to accumulate Cd needs to be enhanced. A better understanding of the genes involved in Cd accumulation in poplar would help to achieve this goal. Here, we monitored the expression of genes known to be involved in Cd uptake, accumulation and translocation from other species, in order to provide information on their potential role in Cd accumulation in poplar. Cd concentration in poplar was significantly higher in roots than in stem and leaves in Cd treated plants. Expression of the poplar homologues of IRT1, NRAMP and OPT3 was initially increased after exposure to Cd but reduced after longer term Cd exposure. Exposure to Cd also influenced the accumulation of Fe, Ca, Cu, Mg and Mn in poplar. In particular, Cd treated plants had a higher concentration of Fe, Ca, Cu, and Mg in leaves and stem compared to control plants after one day and one week of experiment; while in roots after one month Cd treated plants had a lower concentration of Mn, Fe, Cu, Co, and Mg.

Sulphur (S) is an essential element for all living organisms. The uptake, assimilation and metabolism of S in plants are well studied. However, the regulation of S homeostasis remains largely unknown. Here, we report on the identification and characterisation of the more sulphur accumulation1 (msa1-1) mutant. The MSA1 protein is localized to the nucleus and is required for both S-adenosylmethionine (SAM) production and DNA methylation. Loss of function of the nuclear localised MSA1 leads to a reduction in SAM in roots and a strong S-deficiency response even at ample S supply, causing an over-accumulation of sulphate, sulphite, cysteine and glutathione. Supplementation with SAM suppresses this high S phenotype. Furthermore, mutation of MSA1 affects genome-wide DNA methylation, including the methylation of S-deficiency responsive genes. Elevated S accumulation in msa1-1 requires the increased expression of the sulphate transporter genes SULTR1;1 and SULTR1;2 which are also differentially methylated in msa1-1. Our results suggest a novel function for MSA1 in the nucleus in regulating SAM biosynthesis and maintaining S homeostasis epigenetically via DNA methylation.

Supplementary figures. A collection of extra figures which may be of interest to readers but that arenâ t in the main scope of the submission. Figures S1â S4 show step-wise discriminant analyses plots using different subsets of the traits measured (root morphology traits, leaf mineral composition traits, seed mineral composition traits, & seed yield traits respectively). Plots from analyses using the full set of traits are included as a main figure in the submission; see Fig. 7. Figures S5 and S6 are box and whisker plots of seed yield data by crop habit and thousand seed weight by genotype release date respectively. (PPTX 429 kb)

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Understanding the molecular mechanism of adaptive evolution in plants provides insights into the selective forces driving adaptation and the genetic basis of adaptive traits with agricultural value. The genomic resources available for Arabidopsis (Arabidopsis thaliana) make it well suited to the rapid molecular dissection of adaptive processes. Although numerous potentially adaptive loci have been identified in Arabidopsis, the consequences of divergent selection and migration (both important aspects of the process of local adaptation) for Arabidopsis are not well understood. Here, we use a multiyear field-based reciprocal transplant experiment to detect local populations of Arabidopsis composed of multiple small stands of plants (demes) that are locally adapted to the coast and adjacent inland habitats in northeastern Spain. We identify fitness tradeoffs between plants from these different habitats when grown together in inland and coastal common gardens and also, under controlled conditions in soil excavated from coastal and inland sites. Plants from the coastal habitat also outperform those from inland when grown under high salinity, indicating local adaptation to soil salinity. Sodium can be toxic to plants, and we find its concentration to be elevated in soil and plants sampled at the coast. We conclude that the local adaptation that we observe between adjacent coastal and inland populations is caused by ongoing divergent selection driven by the differential salinity between coastal and inland soils.

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.

Genome-wide association (GWA) analyses have generally been used to detect individual loci contributing to the phenotypic diversity in a population by the effects of these loci on the trait mean. More rarely, loci have also been detected based on variance differences between genotypes. Several hypotheses have been proposed to explain the possible genetic mechanisms leading to such variance signals. However, little is known about what causes these signals, or whether this genetic variance-heterogeneity reflects mechanisms of importance in natural populations. Previously, we identified a variance-heterogeneity GWA (vGWA) signal for leaf molybdenum concentrations in Arabidopsis thaliana. Here, fine-mapping of this association reveals that the vGWA emerges from the effects of three independent genetic polymorphisms that all are in strong LD with the markers displaying the genetic variance-heterogeneity. By revealing the genetic architecture underlying this vGWA signal, we uncovered the molecular source of a significant amount of hidden additive genetic variation or “missing heritability”. Two of the three polymorphisms underlying the genetic variance-heterogeneity are promoter variants for Molybdate transporter 1 (MOT1), and the third a variant located ~25 kb downstream of this gene. A fourth independent association was also detected ~600 kb upstream of MOT1. Use of a T-DNA knockout allele highlights Copper Transporter 6; COPT6 (AT2G26975) as a strong candidate gene for this association. Our results show that an extended LD across a complex locus including multiple functional alleles can lead to a variance-heterogeneity between genotypes in natural populations. Further, they provide novel insights into the genetic regulation of ion homeostasis in A. thaliana, and empirically confirm that variance-heterogeneity based GWA methods are a valuable tool to detect novel associations of biological importance in natural populations.

Breeding has transformed wild plant species into modern crops, increasing the allocation of their photosynthetic assimilate into grain, fiber, and other products for human use. Despite progress in increasing the harvest index, much of the biomass of crop plants is not utilized. Potential uses for the large amounts of agricultural residues that accumulate are animal fodder or bioenergy, though these may not be economically viable without additional efforts such as targeted breeding or improved processing. We characterized leaf and stem tissue from a diverse set of rice genotypes (varieties) grown in two environments (greenhouse and field) and report bioenergy-related traits across these variables. Among the 16 traits measured, cellulose, hemicelluloses, lignin, ash, total glucose, and glucose yield changed across environments, irrespective of the genotypes. Stem and leaf tissue composition differed for most traits, consistent with their unique functional contributions and suggesting that they are under separate genetic control. Plant variety had the least influence on the measured traits. High glucose yield was associated with high total glucose and hemicelluloses, but low lignin and ash content. Bioenergy yield of greenhouse-grown biomass was higher than field-grown biomass, suggesting that greenhouse studies overestimate bioenergy potential. Nevertheless, glucose yield in the greenhouse predicts glucose yield in the field (ρ = 0.85, p < 0.01) and could be used to optimize greenhouse (GH) and field breeding trials. Overall, efforts to improve cell wall composition for bioenergy require consideration of production environment, tissue type, and variety.

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.

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.

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.

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.