Phenotypic and genetic dissection of the contents of important metallic elements in hybrid rice grown in cadmium-contaminated paddy fields.

Rice (Oryza sativa L.) is a staple food that feeds over half of the world's population, and the contents of metallic elements in rice grain play important roles in human nutrition. In this study, the contents of important metallic elements were determined by ICP-OES, and included cadmium (Cd), zinc (Zn), manganese (Mn), copper (Cu), iron (Fe), nickel (Ni), calcium (Ca), and magnesium (Mg) in brown rice, in the first node from the top (Node 1), in the second node from the top (Node 2), and in roots of 55 hybrids and their parental lines. The heritability of metallic element contents (MECs), the general combining ability (GCA) for MEC, and the correlation between MECs in different organs/tissues of hybrids were also analyzed. The results indicated that: (1) there was a positive correlation between the contents of Cd and Zn in nodes and roots, but a negative correlation between the contents of Cd and Zn in brown rice of the hybrids(2) the GCA for MECs can be used to evaluate the ability of the parental lines to improve the metal contents in brown rice of the hybrids(3) the contents of Cd, Zn, Ca, and Mg in brown rice were mainly affected by additive genetic effects(4) the restorer lines R2292 and R2265 can be used to cultivate hybrids with high Zn and low Cd contents in the brown rice.

Stress and Internet Addiction: Mediated by Anxiety and Moderated by Self-Control.

Background: The link between stress and Internet addiction (IA) has been confirmed. However, the mechanism underlying the correlation is poorly understood. Thus, the current study proposed a moderated mediation model to test the mediating role of anxiety and the moderating role of self-control (SC) in the link between stress and IA. Methods: Eight hundred and sixty-one Chinese college students (Mage = 20.62 years; SD = 1.58; male = 47.7%) were required to complete an online questionnaire package, including a depression-anxiety-stress scale, a self-control scale, and an Internet addiction test. The PROCESS macro developed based on SPSS was used to test the moderated mediation model. Results: When controlling for gender and age, the results revealed that anxiety partially mediated the link between stress and IA. Specifically, the more stressed college students are, the higher their anxiety level is, and the more likely they are to become addicted to the Internet. Additionally, the direct and indirect links between stress and IA were all moderated by SC. SC buffered the effect of stress on anxiety and anxiety on IA but enhanced stress on IA. Conclusion: These findings emphasized the predictor role of stress on IA and provided insights on intervening in college students' excessive Internet use behaviors for educators, such as reducing anxiety levels and improving self-control abilities.

CF1 reduces grain-cadmium levels in rice (Oryza sativa).

Rice (Oryza sativa) is a leading source of dietary cadmium (Cd), a non-essential heavy metal that poses a serious threat to human health. There are significant variations in grain-Cd levels in natural rice populations, which make the breeding of low-Cd rice a cost-effective way to mitigate grain-Cd accumulation. However, the genetic factors that regulate grain-Cd accumulation have yet to be fully established, thereby hindering the development of low-Cd varieties. Here, we reported a low-Cd quantitative trait locus, CF1, that has the potential to reduce Cd accumulation in rice grains. CF1 is allelic to the metal transporter OsYSL2, which transports Fe from the roots to the shoots. However, it is incapable of binding Cd, and thus, reduces grain-Cd levels indirectly rather than directly in the form of upward delivery. Further analysis showed that high expression levels of CF1 improve Fe nutrition in the shoots, subsequently inhibiting Cd uptake by systemically inhibiting expression of the main Cd uptake gene OsNramp5 in the roots. Compared with the CF1 allele from '02428' (CF102428 ), higher expression levels of CF1 from 'TQ' (CF1TQ ) increased the Fe contents and decreased Cd levels in rice grains. In natural rice populations, CF1TQ was found to be a minor allele, while CF102428 is present in most japonica rice, suggesting that CF1TQ could be widely integrated into the japonica rice genome to generate low-Cd varieties. Overall, these results broaden our mechanistic understanding of the natural variation in grain-Cd accumulation, supporting marker-assisted selection of low-Cd rice.

The Harbinger transposon-derived gene PANDA epigenetically coordinates panicle number and grain size in rice.

Transposons significantly contribute to genome fractions in many plants. Although numerous transposon-related mutations have been identified, the evidence regarding transposon-derived genes regulating crop yield and other agronomic traits is very limited. In this study, we characterized a rice Harbinger transposon-derived gene called PANICLE NUMBER AND GRAIN SIZE (PANDA), which epigenetically coordinates panicle number and grain size. Mutation of PANDA caused reduced panicle number but increased grain size in rice, while transgenic plants overexpressing this gene showed the opposite phenotypic change. The PANDA-encoding protein can bind to the core polycomb repressive complex 2 (PRC2) components OsMSI1 and OsFIE2, and regulates the deposition of H3K27me3 in the target genes, thereby epigenetically repressing their expression. Among the target genes, both OsMADS55 and OsEMF1 were negative regulators of panicle number but positive regulators of grain size, partly explaining the involvement of PANDA in balancing panicle number and grain size. Moreover, moderate overexpression of PANDA driven by its own promoter in the indica rice cultivar can increase grain yield. Thus, our findings present a novel insight into the epigenetic control of rice yield traits by a Harbinger transposon-derived gene and provide its potential application for rice yield improvement.

Genome-wide characterization of i-motifs and their potential roles in the stability and evolution of transposable elements in rice.

I-motifs (iMs) are non-canonical DNA secondary structures that fold from cytosine (C)-rich genomic DNA regions termed putative i-motif forming sequences (PiMFSs). The structure of iMs is stabilized by hemiprotonated C-C base pairs, and their functions are now suspected in key cellular processes in human cells such as genome stability and regulation of gene transcription. In plants, their biological relevance is still largely unknown. Here, we characterized PiMFSs with high potential for i-motif formation in the rice genome by developing and applying a protocol hinging on an iMab antibody-based immunoprecipitation (IP) coupled with high-throughput sequencing (seq), consequently termed iM-IP-seq. We found that PiMFSs had intrinsic subgenomic distributions, cis-regulatory functions and an intricate relationship with DNA methylation. We indeed found that the coordination of PiMFSs with DNA methylation may affect dynamics of transposable elements (TEs) among different cultivated Oryza subpopulations or during evolution of wild rice species. Collectively, our study provides first and unique insights into the biology of iMs in plants, with potential applications in plant biotechnology for improving important agronomic rice traits.

Epigenomic features of DNA G-quadruplexes and their roles in regulating rice gene transcription.

A DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure involved in many biological processes in mammals. The current knowledge on plant DNA G4s, however, is limited; whether and how DNA G4s impact gene expression in plants is still largely unknown. Here, we applied a protocol referred to as BG4-DNA-IP-seq followed by a comprehensive characterization of DNA G4s in rice (Oryza sativa L.); we next integrated dG4s (experimentally detectable G4s) with existing omics data and found that dG4s exhibited differential DNA methylation between transposable element (TE) and non-TE genes. dG4 regions displayed genic-dependent enrichment of epigenomic signatures; finally, we showed that these sites displayed a positive association with expression of DNA G4-containing genes when located at promoters, and a negative association when located in the gene body, suggesting localization-dependent promotional/repressive roles of DNA G4s in regulating gene transcription. This study reveals interrelations between DNA G4s and epigenomic signatures, as well as implicates DNA G4s in modulating gene transcription in rice. Our study provides valuable resources for the functional characterization or bioengineering of some of key DNA G4s in rice.

Robust identification of low-Cd rice varieties by boosting the genotypic effect of grain Cd accumulation in combination with marker-assisted selection.

Rice (Oryza sativa L.), a staple for half of the world's population, usually accumulates high levels of cadmium (Cd) in the grain when planted in the Cd-contaminated paddy fields. Genetic improvements using natural variation of grain-Cd accumulation is the most cost-effective way to mitigate the risk of excess Cd accumulation. However, as a complex trait, grain-Cd accumulation is susceptible to environmental variation, which challenges to characterize the genetic nature and subsequently the stable performance of grain-Cd accumulation. To boost the genetic effect on grain-Cd performance, we established an approach of normalization using the comparative grain-Cd value (CCd) following a contrasting field design. Identification of the genetic locus responsible for CCd variation help us develop a low-grain-Cd variety de novo, named 'Lushansimiao', which had lower grain-Cd levels in a large-scale field test and can produce Cd-safe rice following prolonged irrigations in the field with intermediate levels of Cd pollution. Combined CCd evaluating and low-Cd allelic genotyping, another six varieties were also identified as low-grain-Cd rice. Our study paves the way to efficiently quantify the genetic nature of grain-Cd accumulation in rice, and the stable low-Cd rice varieties will help to mitigate the risk of excess Cd accumulation in rice.

PIN3-mediated auxin transport contributes to blue light-induced adventitious root formation in Arabidopsis.

Adventitious rooting is a heritable quantitative trait that is influenced by multiple endogenous and exogenous factors in plants, and one important environmental factor required for efficient adventitious root formation is light signaling. However, the physiological significance and molecular mechanism of light underlying adventitious root formation are still largely unexplored. Here, we report that blue light-induced adventitious root formation is regulated by PIN-FORMED3 (PIN3)-mediated auxin transport in Arabidopsis. Adventitious root formation is significantly impaired in the loss-of-function mutants of the blue light receptors, PHOTOROPIN1 (PHOT1) and PHOTOROPIN2 (PHOT2), as well as the phototropic transducer, NON-PHOTOTROPIC HYPOCOTYL3 (NPH3). In addition, blue light enhanced the auxin content in the adventitious root, and the pin3 loss-of-function mutant had a reduced adventitious rooting response under blue light compared to the wild type. The PIN3 protein level was higher in plants treated with blue light than in those in darkness, especially in the hypocotyl pericycle, while PIN3-GFP failed to accumulate in nph3 PIN3::PIN3-GFP. Furthermore, the results showed that PIN3 physically interacted with NPH3, a key transducer in phototropic signaling. Taken together, our study demonstrates that blue light induces adventitious root formation through the phototropic signal transducer, NPH3, which regulates adventitious root formation by affecting PIN3-mediated auxin transport.

Overexpression of OsLCT2, a Low-Affinity Cation Transporter Gene, Reduces Cadmium Accumulation in Shoots and Grains of Rice.

Cadmium (Cd)-contaminated rice is a serious issue affecting food safety. Understanding the molecular regulatory mechanisms of Cd accumulation in rice grains is crucial to minimizing Cd concentrations in grains. We identified a member of the low-affinity cation transporter family, OsLCT2 in rice. It was a membrane protein. OsLCT2 was expressed in all tissues of the elongation and maturation zones in roots, with the strongest expression in pericycle and stele cells adjacent to the xylem. When grown in Cd-contaminated paddy soils, rice plants overexpressing OsLCT2 significantly reduced Cd concentrations in the straw and grains. Hydroponic experiment demonstrated its overexpression decreased the rate of Cd translocation from roots to shoots, and reduced Cd concentrations in xylem sap and in shoots of rice. Moreover, its overexpression increased Zn concentrations in roots by up-regulating the expression of OsZIP9, a gene responsible for Zn uptake. Overexpression of OsLCT2 reduces Cd accumulation in rice shoots and grains by limiting the amounts of Cd loaded into the xylem and restricting Cd translocation from roots to shoots of rice. Thus, OsLCT2 is a promising genetic resource to be engineered to reduce Cd accumulation in rice grains.

Characterization and fine mapping of a new dwarf mutant in Brassica napus.

BACKGROUND: Plant height is an important plant characteristic closely related to yield performance of many crops. Reasonable reduction of plant height of crops is beneficial for improving yield and enhancing lodging resistance. RESULTS: In the present study, we described the Brassica napus dwarf mutant bnd2 that was isolated using ethyl methanesulfonate (EMS) mutagenesis. Compared to wild type (WT), bnd2 exhibited reduced height and shorter hypocotyl and petiole leaves. By crossing the bnd2 mutant with the WT strain, we found that the ratio of the mutant to the WT in the F2 population was close to 1:3, indicating that bnd2 is a recessive mutation of a single locus. Following bulked segregant analysis (BSA) by resequencing, BND2 was found to be located in the 13.77-18.08 Mb interval of chromosome A08, with a length of 4.31 Mb. After fine mapping with single nucleotide polymorphism (SNP) and insertion/deletion (InDel) markers, the gene was narrowed to a 140-Kb interval ranging from 15.62 Mb to 15.76 Mb. According to reference genome annotation, there were 27 genes in the interval, of which BnaA08g20960D had an SNP type variation in the intron between the mutant and its parent, which may be the candidate gene corresponding to BND2. The hybrid line derived from a cross between the mutant bnd2 and the commercial cultivar L329 had similar plant height but higher grain yield compared to the commercial cultivar, suggesting that the allele bnd2 is beneficial for hybrid breeding of lodging resistant and high yield rapeseed. CONCLUSION: In this study, we identified a novel dwarf mutant of rapeseed with a new locus, which may be useful for functional analyses of genetic mechanisms of plant architecture and grain yield in rapeseed.

ZINC TRANSPORTER5 and ZINC TRANSPORTER9 Function Synergistically in Zinc/Cadmium Uptake.

The elements Zinc (Zn) and cadmium (Cd) have similar chemical and physical properties, but contrasting physiological effects in higher organisms. In plants, Zn/Cd transport is mediated by various transporter proteins belonging to different families. In this study, we functionally characterized two Zn transporter genes in rice (Oryza sativa), ZINC TRANSPORTER5 (OsZIP5) and ZINC TRANSPORTER9 (OsZIP9), which are tandem duplicates and act synergistically in Zn/Cd uptake. Both genes encode plasma membrane-localized proteins with influx transporter activity. The expression profiles of OsZIP5 and OsZIP9 overlap in the root epidermis and respond to the local Zn status in the root. However, OsZIP9 is also regulated by systemic signals of Zn status from the shoot. OsZIP5 functions redundantly to OsZIP9, but has a relatively weaker effect. Plants with the knockout mutations oszip5, oszip9, or oszip5oszip9 show impaired Zn/Cd uptake. The decreased Zn/Cd levels and growth retardation in the oszip5 mutant are less severe than in the oszip9 mutant. However, the double mutant oszip5oszip9 showed an enhanced Zn deficiency phenotype compared with the single mutants, and few double-knockout plants were able to survive the entire growth cycle without excessive Zn supply. Transgenic plants overexpressing OsZIP9 had markedly enhanced Zn/Cd levels in the aboveground tissues and brown rice. The results of our study fill a gap in current knowledge of Zn uptake and improve our understanding of Zn/Cd accumulation in rice.

Genetic architecture of subspecies divergence in trace mineral accumulation and elemental correlations in the rice grain.

KEY MESSAGE: Genome differentiation has shaped the divergence in element concentration between rice subspecies and contributed to the correlation among trace minerals in the rice grain. The balance between trace minerals in rice, a staple food for more than half of the world's population, is crucial for human health. However, the genetic basis underlying the correlation between trace minerals has not been fully elucidated. To address this issue, we first quantified the concentrations of 11 trace minerals in the grains of a diversity panel of 575 rice cultivars. We found that eight elements were accumulated at significantly different levels between the indica and japonica subspecies, and we also observed significant correlation patterns among a number of elements. Further, using a genome-wide association study, we identified a total of 96 significant association loci (SALs). The differentiation of the major-effect SALs along with the different number of high-concentration alleles present in the two subspecies shaped the different element performance in indica and japonica varieties. Only a few SALs located in clusters and the majority of SALs showed subspecies/subgroup differentiation, indicating that the correlations between elements in the diversity panel were mainly caused by genome differentiation instead of shared genetic basis. The genetic architecture unveiled in this study will facilitate improvement in breeding for trace mineral content.

Author Correction: Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Characterization of functional relationships of R-loops with gene transcription and epigenetic modifications in rice.

We conducted genome-wide identification of R-loops followed by integrative analyses of R-loops with relation to gene expression and epigenetic signatures in the rice genome. We found that the correlation between gene expression levels and profiled R-loop peak levels was dependent on the positions of R-loops within gene structures (hereafter named "genic position"). Both antisense only (ASO)-R-loops and sense/antisense (S/AS)-R-loops sharply peaked around transcription start sites (TSSs), and these peak levels corresponded positively with transcript levels of overlapping genes. In contrast, sense only (SO)-R-loops were generally spread over the coding regions, and their peak levels corresponded inversely to transcript levels of overlapping genes. In addition, integrative analyses of R-loop data with existing RNA-seq, chromatin immunoprecipitation sequencing (ChIP-seq), DNase I hypersensitive sites sequencing (DNase-seq), and whole-genome bisulfite sequencing (WGBS or BS-seq) data revealed interrelationships and intricate connections among R-loops, gene expression, and epigenetic signatures. Experimental validation provided evidence that the demethylation of both DNA and histone marks can influence R-loop peak levels on a genome-wide scale. This is the first study in plants that reveals novel functional aspects of R-loops, their interrelations with epigenetic methylation, and roles in transcriptional regulation.

Variation of a major facilitator superfamily gene contributes to differential cadmium accumulation between rice subspecies.

Cadmium (Cd) accumulation in rice grain poses a serious threat to human health. While several transport systems have been reported, the complicity of rice Cd transport and accumulation indicates the necessity of identifying additional genes, especially those that are responsible for Cd accumulation divergence between indica and japonica rice subspecies. Here, we show that a gene, OsCd1, belonging to the major facilitator superfamily is involved in root Cd uptake and contributes to grain accumulation in rice. Natural variation in OsCd1 with a missense mutation Val449Asp is responsible for the divergence of rice grain Cd accumulation between indica and japonica. Near-isogenic line tests confirm that the indica variety carrying the japonica allele OsCd1V449 can reduce the grain Cd accumulation. Thus, the japonica allele OsCd1V449 may be useful for reducing grain Cd accumulation of indica rice cultivars through breeding.

OsZIP7 functions in xylem loading in roots and inter-vascular transfer in nodes to deliver Zn/Cd to grain in rice.

Rice has lower zinc (Zn) but higher cadmium (Cd) content in grains than other staple crops. Understanding the molecular mechanisms involved in Zn and Cd transportation could benefit homeostatic control, facilitating optimisation of Zn and Cd levels to provide maximum nutrition and safety. In this study, we functionally characterised in planta the rice (Oryza sativa) transporter OsZIP7, which encodes a plasma membrane-localised protein with influx transport activity for both Zn and Cd. OsZIP7 was expressed in parenchyma cells of vascular bundles in roots and nodes. OsZIP7 knockout resulted in retention of Zn and Cd in roots and basal nodes, which hindered their upward delivery to upper nodes and brown rice. And a short-term labelling experiment with the stable 67Zn isotope showed that Zn was distributed toward roots and basal regions and away from leaves in the mutant compared with wild-type rice. Thus, OsZIP7 plays an integral role in xylem loading in roots and inter-vascular transfer in nodes to preferentially deliver Zn and Cd to developing tissues and rice grains.

Natural variation in the HAN1 gene confers chilling tolerance in rice and allowed adaptation to a temperate climate.

Rice (Oryza sativa L.) is a chilling-sensitive staple crop that originated in subtropical regions of Asia. Introduction of the chilling tolerance trait enables the expansion of rice cultivation to temperate regions. Here we report the cloning and characterization of HAN1, a quantitative trait locus (QTL) that confers chilling tolerance on temperate japonica rice. HAN1 encodes an oxidase that catalyzes the conversion of biologically active jasmonoyl-L-isoleucine (JA-Ile) to the inactive form 12-hydroxy-JA-Ile (12OH-JA-Ile) and fine-tunes the JA-mediated chilling response. Natural variants in HAN1 diverged between indica and japonica rice during domestication. A specific allele from temperate japonica rice, which gained a putative MYB cis-element in the promoter of HAN1 during the divergence of the two japonica ecotypes, enhances the chilling tolerance of temperate japonica rice and allows it to adapt to a temperate climate. The results of this study extend our understanding of the northward expansion of rice cultivation and provide a target gene for the improvement of chilling tolerance in rice.

Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield.

Rice grain with excessive cadmium (Cd) is a major source of dietary Cd intake and a serious threat to health for people who consume rice as a staple food. The development of elite rice cultivars with consistently low Cd content is challenging for conventional breeding approaches, and new strategies urgently need to be developed. Here, we report the development of new indica rice lines with low Cd accumulation and no transgenes by knocking out the metal transporter gene OsNramp5 using CRISPR/Cas9 system. Hydroponic culture showed that Cd concentrations in shoots and roots of osnramp5 mutants were dramatically decreased, resulting in rescue of impaired growth in high Cd condition. Cd-contaminated paddy field trials demonstrated that Cd concentration in osnramp5 grains was consistently less than 0.05 mg/kg, in contrast to high Cd concentrations from 0.33 mg/kg to 2.90 mg/kg in grains of Huazhan (the wild-type indica rice). In particular, the plant yield was not significantly affected in osnramp5 mutants. Furthermore, we developed promising hybrid rice lines with extremely low Cd content in grains. Our work supplies a practical approach to developing Cd pollution-safe indica rice cultivars that minimizes Cd contamination risk in grains.

Genetic Diversity, Rather than Cultivar Type, Determines Relative Grain Cd Accumulation in Hybrid Rice.

Cadmium (Cd) is a toxic element, and rice is known to be a leading source of dietary Cd for people who consume rice as their main caloric resource. Hybrid rice has dominated rice production in southern China and has been adopted worldwide. The characteristics of high yield heterosis of rice hybrids makes the public think intuitively that the hybrid rice accumulates more Cd in grain than do inbred cultivars. A detailed understanding of the genetic basis of grain Cd accumulation in hybrids and developing Cd-safe rice are one of the top priorities for hybrid rice breeders at present. In this study, we investigated genetic diversity and grain Cd levels in 617 elite rice hybrids collected from the middle and lower Yangtze River Valley in China and 68 inbred cultivars from around the world. We found that there are large variations in grain Cd accumulation in both the hybrids and their inbred counterparts. However, we found grain Cd levels in the rice hybrids to be similar to the levels in indica rice inbreds, suggesting that the hybrids do not accumulate more Cd than do the inbred rice cultivars. Further analysis revealed that the high heritability of Cd accumulation in the grain and the single indica population structure increases the risk of Cd over-accumulation in hybrid rice. The genetic effects of Cd-related QTLs, which have been identified in related Cd-QTL mapping studies, were also determined in the hybrid rice population. Four QTLs were identified as being associated with the variation in grain Cd levels; three of these loci exhibited obvious indica-japonica differentiations. Our study will provide a better understanding of grain Cd accumulations in hybrid rice, and pave the way toward effective breeding for high-yielding, low grain-Cd hybrids in the future.