A vacuolar transporter plays important roles in zinc and cadmium accumulation in rice grain.

Rice grain is a poor dietary source of zinc (Zn) but the primary source of cadmium (Cd) for humans; however, the molecular mechanisms for their accumulation in rice grain remain incompletely understood. This study functionally characterized a tonoplast-localized transporter, OsMTP1. OsMTP1 was preferentially expressed in the roots, aleurone layer, and embryo of seeds. OsMTP1 knockout decreased Zn concentration in the root cell sap, roots, aleurone layer and embryo, and subsequently increased Zn concentration in shoots and polished rice (endosperm) without yield penalty. OsMTP1 haplotype analysis revealed elite alleles associated with increased Zn level in polished rice, mostly because of the decreased OsMTP1 transcripts. OsMTP1 expression in yeast enhanced Zn tolerance but did not affect that of Cd. While OsMTP1 knockout resulted in decreased uptake, translocation and accumulation of Cd in plant and rice grain, which could be attributed to the indirect effects of altered Zn accumulation. Our results suggest that rice OsMTP1 primarily functions as a tonoplast-localized transporter for sequestrating Zn into vacuole. OsMTP1 knockout elevated Zn concentration but prevented Cd deposition in polished rice without yield penalty. Thus, OsMTP1 is a candidate gene for enhancing Zn level and reducing Cd level in rice grains.

Two metallothionein genes highly expressed in rice nodes are involved in distribution of Zn to the grain.

A rice node is a hub for distribution of mineral elements; however, most genes highly expressed in the node have not been functionally characterized. Transcriptomic analysis of a rice node revealed that two metallothionein genes, OsMT2b and OsMT2c, were highly expressed in the node I. We functionally characterized these genes in terms of gene expression pattern, cellular and subcellular localization, phenotypic analysis of the single and double knockout mutants and metal-binding ability. Both OsMT2b and OsMT2c were mainly and constitutively expressed in the phloem region of enlarged and diffuse vascular bundles in the nodes and of the anther. Knockout of either OsMT2b or OsMT2c increased zinc (Zn) accumulation in the nodes, but decreased Zn distribution to the panicle, resulting in decreased grain yield. A double mutant, osmt2bmt2c, showed further negative effects on the Zn distribution and grain yield. By contrast, knockout of OsMT2b had a small effect on copper (Cu) accumulation. Both OsMT2b and OsMT2c showed binding ability with Zn, whereas only OsMT2b showed binding ability with Cu in yeast. Our results suggest that both OsMT2b and OsMT2c play an important role mainly in the distribution of Zn to grain through chelation and subsequent transport of Zn in the phloem in rice.

Breeding for low cadmium barley by introgression of a Sukkula-like transposable element.

Barley is the fourth most produced cereal crop in the world and one of the major dietary sources of cadmium (Cd), which poses serious threats to human health. Here, we identify a gene that encodes a P-type heavy metal ATPase 3 (HvHMA3) responsible for grain Cd accumulation in barley. HvHMA3 from the high Cd barley variety Haruna Nijo in Japan and the low Cd variety BCS318 in Afghanistan shared 97% identity at the amino acid level. In addition, the HvHMA3 from both varieties showed similar transport activity for Cd and the same subcellular localization at the tonoplast. However, the expression of HvHMA3 was double in BCS318 than in Haruna Nijo. A 3.3-kilobase Sukkula-like transposable element was found to be inserted upstream of the gene in the low Cd variety, which functioned as a promoter and enhanced the expression of HvHMA3. Introgression of this insertion to an elite barley cultivar through backcrossing resulted in decreased Cd accumulation in the grain grown in Cd-contaminated soil without yield penalty. The decreased Cd accumulation resulting from the insertion was also found in some other barley landraces in the world. Our results indicate that insertion of the Sukkula-like transposable element plays an important role in upregulating HvHMA3 expression.

Jasmonic acid alleviates cadmium toxicity in Arabidopsis via suppression of cadmium uptake and translocation.

Jasmonic acid (JA) is thought to be involved in plant responses to cadmium (Cd) stress, but the underlying molecular mechanisms are poorly understood. Here, we show that Cd treatment rapidly induces the expression of genes promoting endogenous JA synthesis, and subsequently increases the JA concentration in Arabidopsis roots. Furthermore, exogenous methyl jasmonate (MeJA) alleviates Cd-generated chlorosis of new leaves by decreasing the Cd concentration in root cell sap and shoot, and decreasing the expression of the AtIRT1, AtHMA2 and AtHMA4 genes promoting Cd uptake and long-distance translocation, respectively. In contrast, mutation of a key JA synthesis gene, AtAOS, greatly enhances the expression of AtIRT1, AtHMA2 and AtHMA4, increases Cd concentration in both roots and shoots, and confers increased sensitivity to Cd. Exogenous MeJA recovers the enhanced Cd-sensitivity of the ataos mutant, but not of atcoi1, a JA receptor mutant. In addition, exogenous MeJA reduces NO levels in Cd-stressed Arabidopsis root tips. Taken together, our results suggest that Cd-induced JA acts via the JA signaling pathway and its effects on NO levels to positively restrict Cd accumulation and alleviates Cd toxicity in Arabidopsis via suppression of the expression of genes promoting Cd uptake and long-distance translocation.

Vascular Cambium-Localized AtSPDT Mediates Xylem-to-Phloem Transfer of Phosphorus for Its Preferential Distribution in Arabidopsis.

During plant growth and development mineral elements are preferentially delivered to different organs and tissues to meet the differential demand. It has been shown that the preferential distribution of mineral nutrients in gramineous plants is mediated by node-based transporters, but the mechanisms of preferential distribution in dicots are poorly understood. Here, we report a distinct mechanism for the preferential distribution of phosphorus (P) in Arabidopsis plants, revealed by detailed functional analysis of AtSPDT/AtSULTR3;4 (SULTR-like P Distribution Transporter), a homolog of rice OsSPDT. Like OsSPDT, AtSPDT is localized at the plasma membrane and showed proton-dependent transport activity for P. Interestingly, we found that AtSPDT is mainly expressed in the rosette basal region and leaf petiole, and its expression is up-regulated by P deficiency. Tissue-specific analysis showed that AtSPDT is mainly located in the vascular cambium of different organs, as well as in the parenchyma tissues of both xylem and phloem regions. Knockout of AtSPDT inhibited the growth of new leaves under low P due to decreased P distribution to those organs. The seed yields of the wild-type and atspdt mutant plants are similar, but the seeds of mutant plants contain - less P. These results indicate that AtSPDT localized in the vascular cambium is involved in preferential distribution of P to the developing tissues, through xylem-to-phloem transfer mainly at the rosette basal region and leaf petiole.

The Putative Peptide Gene FEP1 Regulates Iron Deficiency Response in Arabidopsis.

Iron is an essential element for all organisms, and plants have developed sophisticated systems to acquire iron and maintain iron homeostasis. We found that an Arabidopsis thaliana ABA-hypersensitive mutant, aba hypersensitive germination2-1 (ahg2-1), that is known to be defective in mitochondrial mRNA regulation, had increased expression of iron deficiency response genes. The ahg2-1 mutant had lower heme levels than the wild type. Transcriptome data further revealed that novel genes encoding short polypeptides were highly expressed in this mutant. The expression of one of these genes, which we named FE-UPTAKE-INDUCING PEPTIDE 1 (FEP1), was induced under iron-deficient conditions and was observed in the vascular tissues of the leaves and roots, as well as in leaf mesophyll cells. Notably, deletion or insertion mutations of FEP1 exhibited impaired iron accumulation in shoots but normal iron levels in roots. Artificially induced expression of FEP1 was sufficient to induce iron deficiency response genes, such as basic HELIX-LOOP-HELIX 38 (bHLH38), bHLH39, IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2), and led to iron accumulation in planta. Further analysis confirmed that the encoded peptide, but not the FEP1 RNA, was responsible for this activity. Remarkably, the activation of bHLH39 by FEP1 was independent of FER-LIKE IRON DEFICIENCY INDUCED (FIT), a key transcription factor in the iron deficiency response. Taken together, our results indicate that FEP1 functions in iron homeostasis through a previously undescribed regulatory mechanism for iron acquisition in Arabidopsis.

Two MATE Transporters with Different Subcellular Localization are Involved in Al Tolerance in Buckwheat.

Buckwheat (Fagopyrum esculentum) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms responsible for this high Al tolerance are still poorly understood. Here, we investigated the involvement of two MATE (multi-drug and toxic compound extrusion) genes in Al tolerance. Both FeMATE1 and FeMATE2 showed efflux transport activity for citrate, but not for oxalate when expressed in Xenopus oocytes. A transient assay with buckwheat leaf protoplasts using green fluorescent protein (GFP) fusion showed that FeMATE1 was mainly localized to the plasma membrane, whereas FeMATE2 was localized to the trans-Golgi and Golgi. The expression of FeMATE1 was induced by Al only in the roots, but that of FeMATE2 was up-regulated in both the roots and leaves. Furthermore, the expression of both genes only responded to Al toxicity, but not to other stresses including low pH, cadmium (Cd) and lanthanum (La). Heterologous expression of FeMATE1 or FeMATE2 in the Arabidopsis mutant atmate partially rescued its Al tolerance. Expression of FeMATE1 also partially recovered the Al-induced secretion of citrate in the transgenic lines, whereas expression of FeMATE2 did not complement the citrate secretion. Further physiological analysis showed that buckwheat roots also secreted citrate in addition to oxalate in response to Al in a dose-responsive manner. Taken together, our results indicate that FeMATE1 is involved in the Al-activated citrate secretion in the roots, while FeMATE2 is probably responsible for transporting citrate into the Golgi system for the internal detoxification of Al in the roots and leaves of buckwheat.

Functional characterization of two half-size ABC transporter genes in aluminium-accumulating buckwheat.

Buckwheat (Fagopyrum esculentum Moench) is able to detoxify high aluminium (Al) internally by sequestering it to the vacuoles in the leaves; however, the molecular mechanisms underlying this sequestration are unknown. We performed proteomic analysis with the leaf tonoplast-rich fraction and identified two half-size ABC transporters; FeASL1.1 and FeALS1.2. We investigated the gene expression patterns and subcellular localization. To demonstrate their physiological role, we expressed FeALS1.1 or FeALS1.2 in the Arabidopsis atals1 mutant under the control of AtALS1 promoter. FeALS1.1 expression was upregulated by Al in both the leaves and the roots, and its expression level in the roots was six times higher than its homologous gene (AtALS1) of Arabidopsis. FeALS1.2 expression, however, was not affected by Al but showed a 39 times higher expression level than AtALS1 in the leaves. When FeALS1.1 or FeALS1.2 was expressed in atals1, both of them recovered their Al tolerance through altering the subcellular localization of Al in root cells. Taken together, our results indicate that FeALS1.1 and FeALS1.2 are involved in the internal detoxification of Al in the roots and leaves, respectively, by sequestering Al into the vacuoles. Their high expression is probably required for high Al tolerance in buckwheat.

Abscisic acid alleviates iron deficiency by promoting root iron reutilization and transport from root to shoot in Arabidopsis.

Abscisic acid (ABA) has been demonstrated to be involved in iron (Fe) homeostasis, but the underlying mechanism is largely unknown. Here, we found that Fe deficiency induced ABA accumulation rapidly (within 6 h) in the roots of Arabidopsis. Exogenous ABA at 0.5 µM decreased the amount of root apoplastic Fe bound to pectin and hemicellulose, and increased the shoot Fe content significantly, thus alleviating Fe deficiency-induced chlorosis. Exogenous ABA promoted the secretion of phenolics to release apoplastic Fe and up-regulated the expression of AtNRAMP3 to enhance reutilization of Fe stored in the vacuoles, leading to a higher level of soluble Fe and lower ferric-chelate reductase (FCR) activity in roots. Treatment with ABA also led to increased Fe concentrations in the xylem sap, partially because of the up-regulation of AtFRD3, AtYSL2 and AtNAS1, genes related to long-distance transport of Fe. Exogenous ABA could not alleviate the chlorosis of abi5 mutant resulting from the significantly low expression of AtYSL2 and low transport of Fe from root to shoot. Taken together, our data support the conclusion that ABA is involved in the reutilization and transport of Fe from root to shoot under Fe deficiency conditions in Arabidopsis.

Exogenous auxin alleviates cadmium toxicity in Arabidopsis thaliana by stimulating synthesis of hemicellulose 1 and increasing the cadmium fixation capacity of root cell walls.

Auxin is involved in not only plant physiological and developmental processes but also plant responses to abiotic stresses. In this study, cadmium (Cd(2+)) stress decreased the endogenous auxin level, whereas exogenous auxin (α-naphthaleneacetic acid, NAA, a permeable auxin analog) reduced shoot Cd(2+) concentration and rescued Cd(2+)-induced chlorosis in Arabidopsis thaliana. Under Cd(2+) stress conditions, NAA increased Cd(2+) retention in the roots and most Cd(2+) in the roots was fixed in hemicellulose 1 of the cell wall. NAA treatment did not affect pectin content and its binding capacity for Cd(2+), whereas it significantly increased the content of hemicellulose 1 and the amount of Cd(2+) retained in it. There were highly significant correlations between Cd(2+) concentrations in the root, cell wall and hemicellulose 1 when the plants were subjected to Cd(2+) or NAA+Cd(2+) treatment for 1 to 7d, suggesting that the increase in hemicellulose 1 contributes greatly to the fixation of Cd(2+) in the cell wall. Taken together, these results demonstrate that auxin-induced alleviation of Cd(2+) toxicity in Arabidopsis is mediated through increasing hemicellulose 1 content and Cd(2+) fixation in the root, thus reducing the translocation of Cd(2+) from roots to shoots.

Coordination between apoplastic and symplastic detoxification confers plant aluminum resistance.

Whether aluminum toxicity is an apoplastic or symplastic phenomenon is still a matter of debate. Here, we found that three auxin overproducing mutants, yucca, the recessive mutant superroot2, and superroot1 had increased aluminum sensitivity, while a transfer DNA insertion mutant, xyloglucan endotransglucosylase/hydrolases15 (xth15), showed enhanced aluminum resistance, accompanied by low endogenous indole-3-acetic acid levels, implying that auxin may be involved in plant responses to aluminum stress. We used yucca and xth15 mutants for further study. The two mutants accumulated similar total aluminum in roots and had significantly reduced cell wall aluminum and increased symplastic aluminum content relative to the wild-type ecotype Columbia, indicating that altered aluminum levels in the symplast or cell wall cannot fully explain the differential aluminum resistance of these two mutants. The expression of Al sensitive1 (ALS1), a gene that functions in aluminum redistribution between the cytoplasm and vacuole and contributes to symplastic aluminum detoxification, was less abundant in yucca and more abundant in xth15 than the wild type, consistent with possible ALS1 function conferring altered aluminum sensitivity in the two mutants. Consistent with the idea that xth15 can tolerate more symplastic aluminum because of possible ALS1 targeting to the vacuole, morin staining of yucca root tip sections showed more aluminum accumulation in the cytosol than in the wild type, and xth15 showed reduced morin staining of cytosolic aluminum, even though yucca and xth15 had similar overall symplastic aluminum content. Exogenous application of an active auxin analog, naphthylacetic acid, to the wild type mimicked the aluminum sensitivity and distribution phenotypes of yucca, verifying that auxin may regulate aluminum distribution in cells. Together, these data demonstrate that auxin negatively regulates aluminum tolerance through altering ALS1 expression and aluminum distribution within plant cells, and plants must coordinate exclusion and internal detoxification to reduce aluminum toxicity effectively.

XTH31, encoding an in vitro XEH/XET-active enzyme, regulates aluminum sensitivity by modulating in vivo XET action, cell wall xyloglucan content, and aluminum binding capacity in Arabidopsis.

Xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET) activities, encoded by xyloglucan endotransglucosylase-hydrolase (XTH) genes, are involved in cell wall extension by cutting or cutting and rejoining xyloglucan chains, respectively. However, the physiological significance of this biochemical activity remains incompletely understood. Here, we find that an XTH31 T-DNA insertion mutant, xth31, is more Al resistant than the wild type. XTH31 is bound to the plasma membrane and the encoding gene is expressed in the root elongation zone and in nascent leaves, suggesting a role in cell expansion. XTH31 transcript accumulation is strongly downregulated by Al treatment. XTH31 expression in yeast yields a protein with an in vitro XEH:XET activity ratio of >5000:1. xth31 accumulates significantly less Al in the root apex and cell wall, shows remarkably lower in vivo XET action and extractable XET activity, has a lower xyloglucan content, and exhibits slower elongation. An exogenous supply of xyloglucan significantly ameliorates Al toxicity by reducing Al accumulation in the roots, owing to the formation of an Al-xyloglucan complex in the medium, as verified by an obvious change in chemical shift of (27)Al-NMR. Taken together, the data indicate that XTH31 affects Al sensitivity by modulating cell wall xyloglucan content and Al binding capacity.

Gibberellic acid alleviates cadmium toxicity by reducing nitric oxide accumulation and expression of IRT1 in Arabidopsis thaliana.

Gibberellic acid (GA) is involved in not only plant growth and development but also plant responses to abiotic stresses. Here it was found that treating the plants with GA concentrations from 0.1 to 5 µM for 24 h had no obvious effect on root elongation in the absence of cadmium (Cd), whereas in the presence of Cd2+, GA at 5 µM improved root growth, reduced Cd content and lipid peroxidation in the roots, indicating that GA can partially alleviate Cd toxicity. Cd2+ increased nitric oxide (NO) accumulation in the roots, but GA remarkably reduced it, and suppressed the up-regulation of the expression of IRT1. In contrary, the beneficial effect of GA on alleviating Cd toxicity was not observed in an IRT1 knock-out mutant irt1, suggesting the involvement of IRT1 in Cd2+ absorption. Furthermore, the GA-induced reduction of NO and Cd content can also be partially reversed by the application of a NO donor (S-nitrosoglutathione [GSNO]). Taken all these together, the results showed that GA-alleviated Cd toxicity is mediated through the reduction of the Cd-dependent NO accumulation and expression of Cd2+ uptake related gene-IRT1 in Arabidopsis.

Cell wall polysaccharides are involved in P-deficiency-induced Cd exclusion in Arabidopsis thaliana.

The physiological and molecular mechanisms leading to the competitive interactions between phosphorus (P) and metal elements are a matter of debate. In this study, we found that P deficiency can alleviate cadmium (Cd) toxicity in Arabidopsis thaliana (Col-0). Under P deficiency (-P), less Cd was accumulated in the plants and the root cell walls, indicating the operation of a P-deficiency-induced Cd exclusion mechanism. However, organic acid efflux was similar under -P+Cd and +Cd treatments, suggesting that organic acid efflux is not responsible for the Cd exclusion. Interestingly, P deficiency significantly decreased cell wall polysaccharides (pectin and hemicellulose) contents and pectin methylesterase activity, and decreased the Cd retained by the extracted root cell wall. Therefore, we conclude that the modification of cell wall composition is responsible for the Cd exclusion of the root under P deficiency.