Dynamic interactions between SPX proteins, the ubiquitination machinery, and signalling molecules for stress adaptation at a whole-plant level.

The plant macronutrient phosphorus is a scarce resource and plant-available phosphate is limiting in most soil types. Generally, a gene regulatory module called the phosphate starvation response (PSR) enables efficient phosphate acquisition by roots and translocation to other organs. Plants growing on moderate to nutrient-rich soils need to co-ordinate availability of different nutrients and repress the highly efficient PSR to adjust phosphate acquisition to the availability of other macro- and micronutrients, and in particular nitrogen. PSR repression is mediated by a small family of single SYG1/Pho81/XPR1 (SPX) domain proteins. The SPX domain binds higher order inositol pyrophosphates that signal cellular phosphorus status and modulate SPX protein interaction with PHOSPHATE STARVATION RESPONSE1 (PHR1), the central transcriptional regulator of PSR. Sequestration by SPX repressors restricts PHR1 access to PSR gene promoters. Here we focus on SPX4 that primarily acts in shoots and sequesters many transcription factors other than PHR1 in the cytosol to control processes beyond the classical PSR, such as nitrate, auxin, and jasmonic acid signalling. Unlike SPX1 and SPX2, SPX4 is subject to proteasomal degradation not only by singular E3 ligases, but also by SCF-CRL complexes. Emerging models for these different layers of control and their consequences for plant acclimation to the environment will be discussed.

The plastid-localized lipoamide dehydrogenase 1 is crucial for redox homeostasis, tolerance to arsenic stress and fatty acid biosynthesis in rice.

Soil contamination with arsenic (As) can cause phytotoxicity and reduce crop yield. The mechanisms of As toxicity and tolerance are not fully understood. In this study, we used a forward genetics approach to isolate a rice mutant, ahs1, that exhibits hypersensitivity to both arsenate and arsenite. Through genomic resequencing and complementation tests, we identified OsLPD1 as the causal gene, which encodes a putative lipoamide dehydrogenase. OsLPD1 was expressed in the outer cell layer of roots, root meristem cells, and in the mesophyll and vascular tissues of leaves. Subcellular localization and immunoblot analysis demonstrated that OsLPD1 is localized in the stroma of plastids. In vitro assays showed that OsLPD1 exhibited lipoamide dehydrogenase (LPD) activity, which was strongly inhibited by arsenite, but not by arsenate. The ahs1 and OsLPD1 knockout mutants exhibited significantly reduced NADH/NAD+ and GSH/GSSG ratios, along with increased levels of reactive oxygen species and greater oxidative stress in the roots compared with wild-type (WT) plants under As treatment. Additionally, loss-of-function of OsLPD1 also resulted in decreased fatty acid concentrations in rice grain. Taken together, our finding reveals that OsLPD1 plays an important role for maintaining redox homeostasis, conferring tolerance to arsenic stress, and regulating fatty acid biosynthesis in rice.

Rice chromatin protein OsHMGB1 is involved in phosphate homeostasis and plant growth by affecting chromatin accessibility.

Although phosphorus is one of the most important essential elements for plant growth and development, the epigenetic regulation of inorganic phosphate (Pi) signaling is poorly understood. In this study, we investigated the biological function and mode of action of the high-mobility-group box 1 protein OsHMGB1 in rice (Oryza sativa), using molecular and genetic approaches. We determined that OsHMGB1 expression is induced by Pi starvation and encodes a nucleus-localized protein. Phenotypic analysis of Oshmgb1 mutant and OsHMGB1 overexpression transgenic plants showed that OsHMGB1 positively regulates Pi homeostasis and plant growth. Transcriptome deep sequencing and chromatin immunoprecipitation followed by sequencing indicated that OsHMGB1 regulates the expression of a series of phosphate starvation-responsive (PSR) genes by binding to their promoters. Furthermore, an assay for transposase-accessible chromatin followed by sequencing revealed that OsHMGB1 is involved in maintaining chromatin accessibility. Indeed, OsHMGB1 occupancy positively correlated with genome-wide chromatin accessibility and gene expression levels. Our results demonstrate that OsHMGB1 is a transcriptional facilitator that regulates the expression of a set of PSR genes to maintain Pi homeostasis in rice by increasing the chromatin accessibility, revealing a key epigenetic mechanism that fine-tune plant acclimation responses to Pi-limited environments.

Molecular mechanisms and genetic improvement of low-phosphorus tolerance in rice.

Phosphorus (P) is a macronutrient required for plant growth and reproduction. Orthophosphate (Pi), the preferred P form for plant uptake, is easily fixed in the soil, making it unavailable to plants. Limited phosphate rock resources, low phosphate fertilizer use efficiency and high demands for green agriculture production make it important to clarify the molecular mechanisms underlying plant responses to P deficiency and to improve plant phosphate efficiency in crops. Over the past 20 years, tremendous progress has been made in understanding the regulatory mechanisms of the plant P starvation response. Here, we systematically review current research on the mechanisms of Pi acquisition, transport and distribution from the rhizosphere to the shoot; Pi redistribution and reuse during reproductive growth; and the molecular mechanisms of arbuscular mycorrhizal symbiosis in rice (Oryza sativa L.) under Pi deficiency. Furthermore, we discuss several strategies for boosting P utilization efficiency and yield in rice.

Characterizing membrane anchoring of leaf-form ferredoxin-NADP+ oxidoreductase in rice.

Leaf-form ferredoxin-NADP+ oxidoreductases (LFNRs) function in the last step of the photosynthetic electron transport chain, exist as soluble proteins in the chloroplast stroma and are weakly associated with thylakoids or tightly anchored to chloroplast membranes. Arabidopsis thaliana has two LFNRs, and the chloroplast proteins AtTROL and AtTIC62 participate in anchoring AtLFNRs to the thylakoid membrane. By contrast, the membrane anchoring mechanism of rice (Oryza sativa) LFNRs has not been elucidated. Here, we investigated the membrane-anchoring mechanism of LFNRs and its physiological roles in rice. We characterized the rice protein OsTROL1 based on its homology to AtTROL. We determined that OsTROL1 is also a thylakoid membrane anchor and its loss leads to a compensatory increase in OsTIC62. OsLFNR1 attachment through a membrane anchor depends on OsLFNR2, unlike the Arabidopsis counterparts. In addition, OsTIC62 was more highly expressed in the dark than under light conditions, consistent with the increased membrane binding of OsLFNR in the dark. Moreover, we observed reciprocal stabilization between OsLFNRs and their membrane anchors. In addition, unlike in Arabidopsis, the loss of LFNR membrane anchor affects photosynthesis in rice. Overall, our study sheds light on the mechanisms anchoring LFNRs to membranes in rice and highlights differences with Arabidopsis.

PROTEIN PHOSPHATASE95 Regulates Phosphate Homeostasis by Affecting Phosphate Transporter Trafficking in Rice.

Phosphate (Pi) uptake in plants depends on plasma membrane (PM)-localized phosphate transporters (PTs). OsCK2 phosphorylates PTs and inhibits their trafficking from the endoplasmic reticulum (ER) to the PM in rice (Oryza sativa), but how PTs are dephosphorylated is unknown. We demonstrate that the protein phosphatase type 2C (PP2C) protein phosphatase OsPP95 interacts with OsPT2 and OsPT8 and dephosphorylates OsPT8 at Ser-517. Rice plants overexpressing OsPP95 reduced OsPT8 phosphorylation and promoted OsPT2 and OsPT8 trafficking from the ER to the PM, resulting in Pi accumulation. Under Pi-sufficient conditions, Pi levels were lower in young leaves and higher in old leaves in ospp95 mutants than in those of the wild type, even though the overall shoot Pi levels were the same in the mutant and the wild type. In the wild type, OsPP95 accumulated under Pi starvation but was rapidly degraded under Pi-sufficient conditions. We show that OsPHO2 interacts with and induces the degradation of OsPP95. We conclude that OsPP95, a protein phosphatase negatively regulated by OsPHO2, positively regulates Pi homeostasis and remobilization by dephosphorylating PTs and affecting their trafficking to the PM, a reversible process required for adaptation to variable Pi conditions.

Phenotypes and Molecular Mechanisms Underlying the Root Response to Phosphate Deprivation in Plants.

Phosphorus (P) is an essential macronutrient for plant growth. The roots are the main organ for nutrient and water absorption in plants, and they adapt to low-P soils by altering their architecture for enhancing absorption of inorganic phosphate (Pi). This review summarizes the physiological and molecular mechanisms underlying the developmental responses of roots to Pi starvation, including the primary root, lateral root, root hair, and root growth angle, in the dicot model plant Arabidopsis thaliana and the monocot model plant rice (Oryza sativa). The importance of different root traits and genes for breeding P-efficient roots in rice varieties for Pi-deficient soils are also discussed, which we hope will benefit the genetic improvement of Pi uptake, Pi-use efficiency, and crop yields.

Root-secreted peptide OsPEP1 regulates primary root elongation in rice.

Hormone-like signaling peptides play essential roles in plant growth and development; however, few peptides regulating root development have been identified in rice (Oryza sativa). Here, we combined liquid chromatography-tandem mass spectrometry (LC-MS/MS) with whole-genome in silico screening for root-secreted peptides in rice. We identified the five-amino-acid PEPTIDE 1 (PEP1) encoded by OsPEP1 (LOC_Os11g09560). OsPEP1 was expressed highly in root tissues, especially root cap cells and epidermal cells in the root maturation zone. Exogenous application of PEP1 inhibited primary root growth. Notably, OsPEP1 RNA interference (RNAi) lines had short primary roots with small meristems and short cells in the root elongation zone; furthermore, the short root phenotype of OsPEP1 RNAi plants could be rescued by exogenous application of PEP1. Our transcriptome data further revealed that PEP1 could reprogram the expression of genes in different pathways, including oxidation-reduction. OsPEP1 overexpression lines similarly displayed short roots, although this phenotype was not rescued by exogenous PEP1. These results suggest that root growth can be inhibited by both too much and too little PEP1. Our findings highlight PEP1 as a candidate plant peptide hormone regulating root development in rice.

OsbHLH6 interacts with OsSPX4 and regulates the phosphate starvation response in rice.

Low soil phosphorus (P) availability is a major limitation for crop production. The molecular mechanisms underlying plant responses and adaptation to phosphate (Pi) deficiency are unclear. OsbHLH6 (hereafter bHLH6), an uncharacterized rice (Oryza sativa) Pi starvation response gene encoding a basic helix-loop-helix protein, was identified by yeast two-hybrid screening using the phosphate response repressor OsSPX4 (hereafter SPX4) as bait. bHLH6 is expressed in shoots and roots, and its expression is significantly induced in shoots by Pi deficiency. bHLH6 overexpression lines showed Pi accumulation and enhanced Pi starvation responses, including upregulation of Pi starvation-induced genes and longer root hairs. A bhlh6 mutant showed no significant phenotype variation at the seedling stage. A pull-down assay indicated that bHLH6 had higher binding affinity with SPX4 compared to OsPHR2; therefore, bHLH6 competitively inhibited the interaction of SPX4 and OsPHR2. SPX4 overexpression rescued the Pi accumulation caused by bHLH6 overexpression under high- and low-P conditions. Moreover, overexpression of bHLH6 in an spx4 background did not affect the Pi content of spx4 under high- and low-P conditions. The bhlh6 spx4 double mutant showed lower shoot Pi concentrations and transcript levels of OsPT3 and OsPT10 compared with the spx4 mutant under high-P conditions. RNA sequencing results indicated that bHLH6 overexpression and spx4 mutant lines share many differentially expressed Pi-responsive genes. Therefore, bHLH6 is an important regulator for Pi signaling and homeostasis which antagonizes SPX4. This knowledge helps elucidate the molecular regulation of plant adaptation to Pi deficiency and will promote efforts toward the creation of low Pi-tolerant crops.

OsAMT1;1 and OsAMT1;2 Coordinate Root Morphological and Physiological Responses to Ammonium for Efficient Nitrogen Foraging in Rice.

Optimal plant growth and development rely on morphological and physiological adaptions of the root system to forage heterogeneously distributed nitrogen (N) in soils. Rice grows mainly in the paddy soil where ammonium (NH4+) is present as the major N source. Although root NH4+ foraging behaviors are expected to be agronomically relevant, the underlying mechanism remains largely unknown. Here, we showed that NH4+ supply transiently enhanced the high-affinity NH4+ uptake and stimulated lateral root (LR) branching and elongation. These synergistic physiological and morphological responses were closely related to NH4+-induced expression of NH4+ transporters OsAMT1;1 and OsAMT1;2 in roots. The two independent double mutants (dko) defective in OsAMT1;1 and OsAMT1;2 failed to induce NH4+ uptake and stimulate LR formation, suggesting that OsAMT1s conferred the substrate-dependent root NH4+ foraging. In dko plants, NH4+ was unable to activate the expression of OsPIN2, and the OsPIN2 mutant (lra1) exhibited a strong reduction in NH4+-triggered LR branching, suggesting that the auxin pathway was likely involved in OsAMT1s-dependent LR branching. Importantly, OsAMT1s-dependent root NH4+ foraging behaviors facilitated rice growth and N acquisition under fluctuating NH4+ supply. These results revealed an essential role of OsAMT1s in synergizing root morphological and physiological processes, allowing for efficient root NH4+ foraging to optimize N capture under fluctuating N availabilities.

VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) family proteins are regulators of root system architecture.

Root system architecture (RSA) is a key factor in the efficiency of nutrient capture and water uptake in plants. Understanding the genetic control of RSA will be useful in minimizing fertilizer and water usage in agricultural cropping systems. Using a hydroponic screen and a gel-based imaging system, we identified a rice (Oryza sativa) gene, VAP-RELATED SUPPRESSOR OF TOO MANY MOUTHS1 (OsVST1), which plays a key role in controlling RSA. This gene encodes a homolog of the VAP-RELATED SUPPRESSORS OF TOO MANY MOUTHS (VST) proteins in Arabidopsis (Arabidopsis thaliana), which promote signaling in stomata by mediating plasma membrane-endoplasmic reticulum contacts. OsVST1 mutants have shorter primary roots, decreased root meristem size, and a more compact RSA. We show that the Arabidopsis VST triple mutants have similar phenotypes, with reduced primary root growth and smaller root meristems. Expression of OsVST1 largely complements the short root length and reduced plant height in the Arabidopsis triple mutant, supporting conservation of function between rice and Arabidopsis VST proteins. In a field trial, mutations in OsVST1 did not adversely affect grain yield, suggesting that modulation of this gene could be used as a way to optimize RSA without an inherent yield penalty.

OsSPL3, an SBP-Domain Protein, Regulates Crown Root Development in Rice.

The major root system of cereals consists of crown roots (or adventitious roots), which are important for anchoring plants in the soil and for water and nutrient uptake. However, the molecular basis of crown root formation is largely unknown. Here, we isolated a rice (Oryza sativa) mutant with fewer crown roots, named lower crown root number1 (lcrn1). Map-based cloning revealed that lcrn1 is caused by a mutation of a putative transcription factor-coding gene, O. sativa SQUAMOSA PROMOTER BINDING PROTEIN-LIKE3 (OsSPL3). We demonstrate that the point mutation in lcrn1 perturbs theO. sativa microRNA156 (OsmiR156)-directed cleavage of OsSPL3 transcripts, resulting in the mutant phenotype. Chromatin immunoprecipitation sequencing assays of OsSPL3 binding sites and RNA sequencing of differentially expressed transcripts in lcrn1 further identified potential direct targets of OsSPL3 in basal nodes, including a MADS-box transcription factor, OsMADS50. OsMADS50-overexpressing plants produced fewer crown roots, phenocopying lcrn1, while knocking out OsMADS50 in the lcrn1 background reversed this phenotype. We also show that OsSPL12, another OsmiR156 target gene, regulates OsMADS50 and crown root development. Taken together, our findings suggest a novel regulatory pathway in which the OsmiR156-OsSPL3/OsSPL12 module directly activates OsMADS50 in the node to regulate crown root development in rice.

Progress on methods for acquiring flanking genomic sequence.

Flanking genomic sequences refer to the DNA sequences flanking specific sites of known sequences in chromosome, which contain information such as candidate genes, transcriptional regulation, chromosome structure, and biosafety, and play an important role in genomics research. Flanking sequence acquisition technologies are mainly used in the cloning of regulatory sequences such as promoters and enhancers, identification of T-DNA or transposon insertion sites, chromosome walking, genome-wide gap filling, etc. It is an important means of structural genomics research and functional genomics research. It is applied in the identification of transgenic plants and animals and their safety management. With the development of molecular biology, many methods for obtaining flanking sequences have been established, including plasmid rescue, inverse PCR, ligation-mediated PCR, semi-random primer PCR, whole-genome resequencing etc. In this review, we summarize and compared different methods for acquiring flanking genomic sequence. The principles and research progress of each approach are discussed.

Phosphate Uptake and Transport in Plants: An Elaborate Regulatory System.

Phosphorus (P) is an essential macronutrient for plant growth and development. Low inorganic phosphate (Pi) availability is a limiting factor for plant growth and yield. To cope with a complex and changing environment, plants have evolved elaborate mechanisms for regulating Pi uptake and use. Recently, the molecular mechanisms of plant Pi signaling have become clearer. Plants absorb Pi from the soil through their roots and transfer Pi to various organs or tissues through phosphate transporters, which are precisely controlled at the transcript and protein levels. Here, we summarize recent progress on the molecular regulatory mechanism of phosphate transporters in Arabidopsis and rice, including the characterization of functional transporters, regulation of transcript levels, protein localization and turnover of phosphate transporters. A more in-depth understanding of plant adaptation to a changing Pi environment will facilitate the genetic improvement of plant P efficiency.

Cadmium Inhibits Lateral Root Emergence in Rice by Disrupting OsPIN-Mediated Auxin Distribution and the Protective Effect of OsHMA3.

Cadmium (Cd) strongly inhibits root growth, especially the formation of lateral roots (LRs). The mechanism of Cd inhibition on LR formation in rice (Oryza sativa) remains unclear. In this study, we found that LR emergence in rice was inhibited significantly by 1 �M Cd and almost completely arrested by 5 �M Cd. Cd suppressed both the formation and subsequent development of the lateral root primordium (LRP). By using transgenic rice expressing the auxin response reporters DR5::GUS and DR5rev::VENUS, we found that Cd markedly reduced the auxin levels in the stele and LRP. Cd rapidly downregulated the expression of the auxin efflux transporter genes OsPIN1b, OsPIN1c and OsPIN9 in the stele and LRP. The emergence of LRs in a rice cultivar with a null allele of OsHMA3 (Heavy Metal ATPase 3) was more sensitive to Cd than cultivars with functional alleles. Overexpression of functional OsHMA3 in rice greatly alleviated the inhibitory effect of Cd, but the protective effect of OsHMA3 was abolished by the auxin polar transport inhibitor 1-N-naphthylphthalamic acid. The results suggest that Cd inhibits LR development in rice by disrupting OsPIN-mediated auxin distribution to LRP and OsHMA3 protects against Cd toxicity by sequestering Cd into the vacuoles.

Rice NIN-LIKE PROTEIN 4 plays a pivotal role in nitrogen use efficiency.

Nitrogen (N) is one of the key essential macronutrients that affects rice growth and yield. Inorganic N fertilizers are excessively used to boost yield and generate serious collateral environmental pollution. Therefore, improving crop N use efficiency (NUE) is highly desirable and has been a major endeavour in crop improvement. However, only a few regulators have been identified that can be used to improve NUE in rice to date. Here we show that the rice NIN-like protein 4 (OsNLP4) significantly improves the rice NUE and yield. Field trials consistently showed that loss-of-OsNLP4 dramatically reduced yield and NUE compared with wild type under different N regimes. In contrast, the OsNLP4 overexpression lines remarkably increased yield by 30% and NUE by 47% under moderate N level compared with wild type. Transcriptomic analyses revealed that OsNLP4 orchestrates the expression of a majority of known N uptake, assimilation and signalling genes by directly binding to the nitrate-responsive cis-element in their promoters to regulate their expression. Moreover, overexpression of OsNLP4 can recover the phenotype of Arabidopsis nlp7 mutant and enhance its biomass. Our results demonstrate that OsNLP4 plays a pivotal role in rice NUE and sheds light on crop NUE improvement.

OsPIN9, an auxin efflux carrier, is required for the regulation of rice tiller bud outgrowth by ammonium.

The degree of rice tillering is an important agronomic trait that can be markedly affected by nitrogen supply. However, less is known about how nitrogen-regulated rice tillering is related to polar auxin transport. Compared with nitrate, ammonium induced tiller development and was paralleled with increased 3 H-indole-acetic acid (IAA) transport and greater auxin into the junctions. OsPIN9, an auxin efflux carrier, was selected as the candidate gene involved in ammonium-regulated tillering based on GeneChip data. Compared with wild-type plants, ospin9 mutants had fewer tillers, and OsPIN9 overexpression increased the tiller number. Additionally, OsPIN9 was mainly expressed in vascular tissue of the junction and tiller buds, and encoded a membrane-localised protein. Heterologous expression in Xenopus oocytes and yeast demonstrated that OsPIN9 is a functional auxin efflux transporter. More importantly, its RNA and protein levels were induced by ammonium but not by nitrate, and tiller numbers in mutants did not respond to nitrogen forms. Further advantages, including increased tiller number and grain yield, were observed in overexpression lines grown in the paddy field at a low-nitrogen rate compared with at a high-nitrogen rate. Our data revealed that ammonium supply and an auxin efflux transporter co-ordinately control tiller bud elongation in rice.

OsbHLH98 regulates leaf angle in rice through transcriptional repression of OsBUL1.

Leaf angle is an important agronomic trait in cereals that helps determine plant yield by affecting planting density. However, the regulation mechanism of leaf angle remained elusive. Here, we show that OsbHLH98, a rice bHLH transcription factor, negatively regulates leaf angle. osbhlh98 mutant leaves formed a larger leaf angle, whereas transgenic plants overexpressing OsbHLH98 exhibited a slight reduction in leaf angle. We determined that the changes in leaf angle resulted from increased number and size of parenchyma cells on the adaxial side of the lamina joint in osbhlh98 mutants. Experiments using reporter constructs showed that OsbHLH98 is expressed on the adaxial side of lamina joints, consistent with its proposed function in regulating leaf angle. Furthermore, we established by chromatin immunoprecipitation and CUT&RUN that OsBUL1 is a direct downstream target of OsbHLH98. Transactivation assays and reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis indicated that OsbHLH98 represses OsBUL1 transcription. Our results demonstrate that OsbHLH98 negatively regulates leaf angle by counteracting brassinosteroid-induced cell elongation via the repression of OsBUL1 transcription. The characterization of OsbHLH98 and its role in determining leaf angle will lay the foundation to develop the ideal plant architecture for adaptation to high planting density.

CASEIN KINASE2-Dependent Phosphorylation of PHOSPHATE2 Fine-tunes Phosphate Homeostasis in Rice.

Plants have evolved complex physiological and biochemical mechanisms to adapt to a heterogeneous soil phosphorus environment. PHOSPHATE2 (PHO2) is a phosphate (Pi) starvation-signaling regulator involved in maintaining Pi homeostasis in plants. Arabidopsis (Arabidopsis thaliana) PHO2 targets PHOSPHATE TRANSPORTER1 (PHT1) and PHO1 for degradation, whereas rice (Oryza sativa) PHO2 is thought to mediate PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 degradation. However, it is unclear whether and how PHO2 is post-translationally regulated. Here, we show that in rice, the CASEIN KINASE2 (OsCK2) catalytic subunit OsCK2α3 interacts with OsPHO2 in vitro and in vivo in vascular tissues cells, and phosphorylates OsPHO2 at Ser-841. Phosphorylated OsPHO2 is degraded more rapidly than native OsPHO2 in cell-free degradation assays. OsPHO2 interacts with OsPHO1 and targets it for degradation through a multivesicular body-mediated pathway. PHO1 mutation partially rescued the pho2 mutant phenotype. Further genetic analysis showed that a nonphosphorylatable version of OsPHO2 rescued the Ospho2 phenotype of high Pi accumulation in leaves better than native OsPHO2. In addition to the previously established role of OsCK2 in negatively regulating endoplasmic reticulum exit of PHT1 phosphate transporters, this work uncovers a role for OsCK2α3 in modulating Pi homeostasis through regulating the phosphorylation status and abundance of OsPHO2 in rice.

Lateral roots but not root hairs contribute to high uptake of manganese and cadmium in rice.

Rice (Oryza sativa L.) can accumulate high manganese (Mn) in the shoots through uptake by the roots, which consist of crown roots, lateral roots and root hairs. We investigated the role of lateral roots and root hairs in Mn and cadmium (Cd) uptake by using two indica rice mutants defective in formation of lateral roots (osiaa11) and root hairs (osrhl1). The uptake of Mn and Cd in osiaa11 was significantly lower than that in wild type 'Kasalath', but there was no difference between wild type and osrhl1. Furthermore, a kinetic study showed that Mn uptake in osiaa11 was much lower than that in wild type and osrhl1 across a wide range of Mn concentrations. The role of lateral roots in Mn and Cd uptake was further confirmed in a japonica rice mutant defective in lateral root formation. We found that expression of Mn transporter gene Natural Resistance-Associated Macrophage Protein 5 (OsNRAMP5), but not of Metal Tolerance Protein 9 (OsMTP9), was lower in osiaa11 than in wild type; however, there were no differences between osrhl1 and the wild type. Immunostaining showed that OsNRAMP5 and OsMTP9 were localized in the exodermis and endodermis of crown roots and lateral roots, but not in the root hairs. Taken together, our results indicate that lateral roots, but not root hairs, play an important role in high Mn and Cd uptake in rice.