More sustainable vegetable oil: Balancing productivity with carbon storage opportunities.

Intensive cultivation and post-harvest vegetable oil production stages are major sources of greenhouse gas (GHG) emissions. Variation between production systems and reporting disparity have resulted in discordance in previous emissions estimates. The aim of this study was to assess global systems-wide variation in GHG emissions resulting from palm, soybean, rapeseed and sunflower oil production. Such an analysis is critical to understand the implications of meeting increasing edible oil demand. To achieve this, we performed a unified re-analysis of life cycle input data from diverse palm, soybean, rapeseed, and sunflower oil production systems, from a saturating search of published literature. The resulting dataset reflects almost 6000 producers in 38 countries, and is representative of over 71% of global vegetable oil production. Across all oil crop systems, median GHG emissions were 3.81 kg CO2e per kg refined oil. Crop specific median emissions ranged from 2.49 kg CO2e for rapeseed oil to 4.25 kg CO2e for soybean oil per kg refined oil. Determination of the carbon cost of agricultural land occupation revealed that carbon storage potential in native compared to agricultural land cover drives variation in production GHG emissions, and indicates that expansion of production in low carbon storage potential land, whilst reforesting areas of high carbon storage potential, could reduce net GHG emissions whilst boosting productivity. Nevertheless, there remains considerable scope to improve sustainability within current production systems, including through increasing yields whilst limiting application of inputs with high carbon footprints, and in the case of palm oil through more widespread adoption of methane capture technologies in processing stages.

Suberin plasticity to developmental and exogenous cues is regulated by a set of MYB transcription factors.

Suberin is a hydrophobic biopolymer that can be deposited at the periphery of cells, forming protective barriers against biotic and abiotic stress. In roots, suberin forms lamellae at the periphery of endodermal cells where it plays crucial roles in the control of water and mineral transport. Suberin formation is highly regulated by developmental and environmental cues. However, the mechanisms controlling its spatiotemporal regulation are poorly understood. Here, we show that endodermal suberin is regulated independently by developmental and exogenous signals to fine-tune suberin deposition in roots. We found a set of four MYB transcription factors (MYB41, MYB53, MYB92, and MYB93), each of which is individually regulated by these two signals and is sufficient to promote endodermal suberin. Mutation of these four transcription factors simultaneously through genome editing leads to a dramatic reduction in suberin formation in response to both developmental and environmental signals. Most suberin mutants analyzed at physiological levels are also affected in another endodermal barrier made of lignin (Casparian strips) through a compensatory mechanism. Through the functional analysis of these four MYBs, we generated plants allowing unbiased investigation of endodermal suberin function, without accounting for confounding effects due to Casparian strip defects, and were able to unravel specific roles of suberin in nutrient homeostasis.

Parallel adaptation in autopolyploid Arabidopsis arenosa is dominated by repeated recruitment of shared alleles.

Relative contributions of pre-existing vs de novo genomic variation to adaptation are poorly understood, especially in polyploid organisms. We assess this in high resolution using autotetraploid Arabidopsis arenosa, which repeatedly adapted to toxic serpentine soils that exhibit skewed elemental profiles. Leveraging a fivefold replicated serpentine invasion, we assess selection on SNPs and structural variants (TEs) in 78 resequenced individuals and discover significant parallelism in candidate genes involved in ion homeostasis. We further model parallel selection and infer repeated sweeps on a shared pool of variants in nearly all these loci, supporting theoretical expectations. A single striking exception is represented by TWO PORE CHANNEL 1, which exhibits convergent evolution from independent de novo mutations at an identical, otherwise conserved site at the calcium channel selectivity gate. Taken together, this suggests that polyploid populations can rapidly adapt to environmental extremes, calling on both pre-existing variation and novel polymorphisms.

Mutation in OsCADT1 enhances cadmium tolerance and enriches selenium in rice grain.

How cadmium (Cd) tolerance in rice is regulated remains poorly understood. We used a forward genetic approach to investigate Cd tolerance in rice. Using a root elongation assay, we isolated a rice mutant with enhanced Cd tolerance, cadt1, from an ethyl methanesulphonate (EMS)-mutagenized population of a widely grown Indica cultivar. The mutant accumulated more Cd in roots but not in shoots and grains. Using genomic resequencing and complementation, we identified OsCADT1 as the causal gene for the mutant phenotype, which encodes a putative serine hydroxymethyltransferase. OsCADT1 protein was localized to the nucleus and the OsCADT1 gene was expressed in both roots and shoots. OsCADT1 mutation resulted in higher sulphur and selenium accumulation in the shoots and grains. Selenate influx in cadt1 was 2.4 times that of the wild-type. The mutant showed higher expression of the sulphate/selenate transporter gene OsSULTR1;1 and the sulphur-deficiency-inducible gene OsSDI1. Thiol compounds including cysteine, glutathione and phytochelatins were significantly increased in the mutant, underlying its increased Cd tolerance. Growth and grain biomass were little affected. The results suggest that OsCADT1 acts as a negative regulator of sulphate/selenate uptake and assimilation. OsCADT1 mutation increases Cd tolerance and enriches selenium in rice grains, providing a novel solution for selenium biofortification.

Variation in the BrHMA3 coding region controls natural variation in cadmium accumulation in Brassica rapa vegetables.

Brassica rapa includes several important leafy vegetable crops with the potential for high cadmium (Cd) accumulation, posing a risk to human health. This study aims to understand the genetic basis underlying the variation in Cd accumulation among B. rapa vegetables. Cd uptake and translocation in 64 B. rapa accessions were compared. The role of the heavy metal ATPase gene BrHMA3 in the variation of Cd accumulation was investigated. BrHMA3 encodes a tonoplast-localized Cd transporter. Five full-length and four truncated haplotypes of the BrHMA3 coding sequence were identified, explaining >80% of the variation in the Cd root to shoot translocation among the 64 accessions and in F2 progeny. Truncated BrHMA3 haplotypes had a 2.3 and 9.3 times higher shoot Cd concentration and Cd translocation ratio, respectively, than full-length haplotypes. When expressed in yeast and Arabidopsis thaliana, full-length BrHMA3 showed activity consistent with a Cd transport function, whereas truncated BrHMA3 did not. Variation in the BrHMA3 promoter sequence had little effect on Cd translocation. Variation in the BrHMA3 coding sequence is a key determinant of Cd translocation to and accumulation in the leaves of B. rapa. Strong alleles of BrHMA3 can be used to breed for B. rapa vegetables that are low in Cd in their edible portions.

Epigenetic regulation of sulfur homeostasis in plants.

Plants have evolved sophisticated mechanisms for adaptation to fluctuating availability of nutrients in soil. Such mechanisms are of importance for plants to maintain homeostasis of nutrient elements for their development and growth. The molecular mechanisms controlling the homeostasis of nutrient elements at the genetic level have been gradually revealed, including the identification of regulatory factors and transporters responding to nutrient stresses. Recent studies have suggested that such responses are controlled not only by genetic regulation but also by epigenetic regulation. In this review, we present recent studies on the involvement of DNA methylation, histone modifications, and non-coding RNA-mediated gene silencing in the regulation of sulfur homeostasis and the response to sulfur deficiency. We also discuss the potential effect of sulfur-containing metabolites such as S-adenosylmethionine on the maintenance of DNA and histone methylation.

Three Genes Define a Bacterial-Like Arsenic Tolerance Mechanism in the Arsenic Hyperaccumulating Fern Pteris vittata.

Arsenic is a carcinogenic contaminant of water and food and a growing threat to human health in many regions of the world. This study focuses on the fern Pteris vittata (Pteridaceae), which is extraordinary in its ability to tolerate and hyperaccumulate very high levels of arsenic that would kill any other plant or animal outside the Pteridaceae. Here, we use RNA-seq to identify three genes (GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE (PvGAPC1), ORGANIC CATION TRANSPORTER 4 (PvOCT4), and GLUTATHIONE S-TRANSFERASE (PvGSTF1) that are highly upregulated by arsenic and are necessary for arsenic tolerance, as demonstrated by RNAi. The proteins encoded by these genes have unexpected properties: PvGAPC1 has an unusual active site and a much greater affinity for arsenate than phosphate; PvGSTF1 has arsenate reductase activity; and PvOCT4 localizes as puncta in the cytoplasm. Surprisingly, PvGAPC1, PvGSTF1, and arsenate localize in a similar pattern. These results are consistent with a model that describes the fate of arsenate once it enters the cell. It involves the conversion of arsenate into 1-arseno-3-phosphoglycerate by PvGAPC1. This "chemically trapped" arsenate is pumped into specific arsenic metabolizing vesicles by the PvOCT4 protein. Once inside these vesicles, 1-arseno-3-phosphoglycerate hydrolyses to release arsenate, which is then reduced by PvGSTF1 to arsenite, the form of arsenic stored in the vacuoles of this fern. This mechanism is strikingly similar to one recently described Pseudomonas aeruginosa, whose tolerance to arsenic also involves the biosynthesis and transport of 1-arseno-3-phosphoglycerate, indicating that P. vittata has evolved a simple, bacterial-like mechanism for arsenic tolerance.

Dissecting the components controlling root-to-shoot arsenic translocation in Arabidopsis thaliana.

Arsenic (As) is an important environmental and food-chain toxin. We investigated the key components controlling As accumulation and tolerance in Arabidopsis thaliana. We tested the effects of different combinations of gene knockout, including arsenate reductase (HAC1), γ-glutamyl-cysteine synthetase (γ-ECS), phytochelatin synthase (PCS1) and phosphate effluxer (PHO1), and the heterologous expression of the As-hyperaccumulator Pteris vittata arsenite efflux (PvACR3), on As tolerance, accumulation, translocation and speciation in A. thaliana. Heterologous expression of PvACR3 markedly increased As tolerance and root-to-shoot As translocation in A. thaliana, with PvACR3 being localized to the plasma membrane. Combining PvACR3 expression with HAC1 mutation led to As hyperaccumulation in the shoots, whereas combining HAC1 and PHO1 mutation decreased As accumulation. Mutants of γ-ECS and PCS1 were hypersensitive to As and had higher root-to-shoot As translocation. Combining γ-ECS or PCS1 with HAC1 mutation did not alter As tolerance or accumulation beyond the levels observed in the single mutants. PvACR3 and HAC1 have large effects on root-to-shoot As translocation. Arsenic hyperaccumulation can be engineered in A. thaliana by knocking out the HAC1 gene and expressing PvACR3. PvACR3 and HAC1 also affect As tolerance, but not to the extent of γ-ECS and PCS1.

A new vesicle trafficking regulator CTL1 plays a crucial role in ion homeostasis.

Ion homeostasis is essential for plant growth and environmental adaptation, and maintaining ion homeostasis requires the precise regulation of various ion transporters, as well as correct root patterning. However, the mechanisms underlying these processes remain largely elusive. Here, we reported that a choline transporter gene, CTL1, controls ionome homeostasis by regulating the secretory trafficking of proteins required for plasmodesmata (PD) development, as well as the transport of some ion transporters. Map-based cloning studies revealed that CTL1 mutations alter the ion profile of Arabidopsis thaliana. We found that the phenotypes associated with these mutations are caused by a combination of PD defects and ion transporter misregulation. We also established that CTL1 is involved in regulating vesicle trafficking and is thus required for the trafficking of proteins essential for ion transport and PD development. Characterizing choline transporter-like 1 (CTL1) as a new regulator of protein sorting may enable researchers to understand not only ion homeostasis in plants but also vesicle trafficking in general.

BRUTUS and its paralogs, BTS LIKE1 and BTS LIKE2, encode important negative regulators of the iron deficiency response in Arabidopsis thaliana.

Iron (Fe) is required for plant health, but it can also be toxic when present in excess. Therefore, Fe levels must be tightly controlled. The Arabidopsis thaliana E3 ligase BRUTUS (BTS) is involved in the negative regulation of the Fe deficiency response and we show here that the two A. thaliana BTS paralogs, BTS LIKE1 (BTSL1) and BTS LIKE2 (BTSL2) encode proteins that act redundantly as negative regulators of the Fe deficiency response. Loss of both of these E3 ligases enhances tolerance to Fe deficiency. We further generated a triple mutant with loss of both BTS paralogs and a partial loss of BTS expression that exhibits even greater tolerance to Fe-deficient conditions and increased Fe accumulation without any resulting Fe toxicity effects. Finally, we identified a mutant carrying a novel missense mutation of BTS that exhibits an Fe deficiency response in the root when grown under both Fe-deficient and Fe-sufficient conditions, leading to Fe toxicity when plants are grown under Fe-sufficient conditions.

Nuclear Localised MORE SULPHUR ACCUMULATION1 Epigenetically Regulates Sulphur Homeostasis in Arabidopsis thaliana.

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.

A heavy metal P-type ATPase OsHMA4 prevents copper accumulation in rice grain.

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.

A loss-of-function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars.

Excessive cadmium (Cd) accumulation in rice poses a risk to food safety. OsHMA3 plays an important role in restricting Cd translocation from roots to shoots. A non-functional allele of OsHMA3 has been reported in some Indica rice cultivars with high Cd accumulation, but it is not known if OsHMA3 allelic variation is associated with Cd accumulation in Japonica cultivars. In this study, we identified a Japonica cultivar with consistently high Cd accumulation in shoots and grain in both field and greenhouse experiments. The cultivar possesses an OsHMA3 allele with a predicted amino acid mutation at the 380(th) position from Ser to Arg. The haplotype had no Cd transport activity when the gene was expressed in yeast, and the allele did not complement a known nonfunctional allele of OsHMA3 in F1 test. The allele is present only in temperate Japonica cultivars among diversity panels of 1483 rice cultivars. Different cultivars possessing this allele showed greatly increased root-to-shoot Cd translocation and a shift in root Cd speciation from Cd-S to Cd-O bonding determined by synchrotron X-ray absorption spectroscopy. Our study has identified a new loss-of-function allele of OsHMA3 in Japonica rice cultivars leading to high Cd accumulation in shoots and grain.

Genome-wide association mapping identifies a new arsenate reductase enzyme critical for limiting arsenic accumulation in plants.

Inorganic arsenic is a carcinogen, and its ingestion through foods such as rice presents a significant risk to human health. Plants chemically reduce arsenate to arsenite. Using genome-wide association (GWA) mapping of loci controlling natural variation in arsenic accumulation in Arabidopsis thaliana allowed us to identify the arsenate reductase required for this reduction, which we named High Arsenic Content 1 (HAC1). Complementation verified the identity of HAC1, and expression in Escherichia coli lacking a functional arsenate reductase confirmed the arsenate reductase activity of HAC1. The HAC1 protein accumulates in the epidermis, the outer cell layer of the root, and also in the pericycle cells surrounding the central vascular tissue. Plants lacking HAC1 lose their ability to efflux arsenite from roots, leading to both increased transport of arsenic into the central vascular tissue and on into the shoot. HAC1 therefore functions to reduce arsenate to arsenite in the outer cell layer of the root, facilitating efflux of arsenic as arsenite back into the soil to limit both its accumulation in the root and transport to the shoot. Arsenate reduction by HAC1 in the pericycle may play a role in limiting arsenic loading into the xylem. Loss of HAC1-encoded arsenic reduction leads to a significant increase in arsenic accumulation in shoots, causing an increased sensitivity to arsenate toxicity. We also confirmed the previous observation that the ACR2 arsenate reductase in A. thaliana plays no detectable role in arsenic metabolism. Furthermore, ACR2 does not interact epistatically with HAC1, since arsenic metabolism in the acr2 hac1 double mutant is disrupted in an identical manner to that described for the hac1 single mutant. Our identification of HAC1 and its associated natural variation provides an important new resource for the development of low arsenic-containing food such as rice.

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.

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.

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

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.

Polyploids exhibit higher potassium uptake and salinity tolerance in Arabidopsis.

Genome duplication (or polyploidization) has occurred throughout plant evolutionary history and is thought to have driven the adaptive radiation of plants. We found that the cytotype of the root, and not the genotype, determined the majority of heritable natural variation in leaf potassium (K) concentration in Arabidopsis thaliana. Autopolyploidy also provided resistance to salinity and may represent an adaptive outcome of the enhanced K accumulation of plants with higher ploidy.

The CTR/COPT-dependent copper uptake and SPL7-dependent copper deficiency responses are required for basal cadmium tolerance in A. thaliana.

Copper (Cu) homeostasis in plants is maintained by at least two mechanisms: (1) the miRNA-dependent reallocation of intracellular Cu among major Cu-enzymes and important energy-related functions; (2) the regulation of the expression of Cu transporters including members of the CTR/COPT family. These events are controlled by the transcription factor SPL7 in Arabidopsis thaliana. Cadmium (Cd), on the other hand, is a non-essential and a highly toxic metal that interferes with homeostasis of essential elements by competing for cellular binding sites. Whether Cd affects Cu homeostasis in plants is unknown. We found that Cd stimulates Cu accumulation in roots of A. thaliana and increases mRNA expression of three plasma membrane-localized Cu uptake transporters, COPT1, COPT2 and COPT6. Further analysis of Cd sensitivity of single and triple copt1copt2copt6 mutants, and transgenic plants ectopically expressing COPT6 suggested that Cu uptake is an essential component of Cd resistance in A. thaliana. Analysis of the contribution of the SPL7-dependent pathway to Cd-induced expression of COPT1, COPT2 and COPT6 showed that it occurs, in part, through mimicking the SPL7-dependent transcriptional Cu deficiency response. This response also involves components of the Cu reallocation system, miRNA398, FSD1, CSD1 and CSD2. Furthermore, seedlings of the spl7-1 mutant accumulate up to 2-fold less Cu in roots than the wild-type, are hypersensitive to Cd, and are more sensitive to Cd than the triple copt1copt2copt6 mutant. Together these data show that exposure to excess Cd triggers SPL7-dependent Cu deficiency responses that include Cu uptake and reallocation that are required for basal Cd tolerance in A. thaliana.

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

BACKGROUND: To 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. RESULTS: We 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. CONCLUSIONS: Here, 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.

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.