Natural variation of TBR confers plant zinc toxicity tolerance through root cell wall pectin methylesterification.

Zinc (Zn) is an essential micronutrient but can be cytotoxic when present in excess. Plants have evolved mechanisms to tolerate Zn toxicity. To identify genetic loci responsible for natural variation of plant tolerance to Zn toxicity, we conduct genome-wide association studies for root growth responses to high Zn and identify 21 significant associated loci. Among these loci, we identify Trichome Birefringence (TBR) allelic variation determining root growth variation in high Zn conditions. Natural alleles of TBR determine TBR transcript and protein levels which affect pectin methylesterification in root cell walls. Together with previously published data showing that pectin methylesterification increase goes along with decreased Zn binding to cell walls in TBR mutants, our findings lead to a model in which TBR allelic variation enables Zn tolerance through modulating root cell wall pectin methylesterification. The role of TBR in Zn tolerance is conserved across dicot and monocot plant species.

Physiological and molecular mechanisms of plant-root responses to iron toxicity.

The chemical form and physiological activity of iron (Fe) in soil are dependent on soil pH and redox potential (Eh), and Fe levels in soils are frequently elevated to the point of causing Fe toxicity in plants, with inhibition of normal physiological activities and of growth and development. In this review, we describe how iron toxicity triggers important physiological changes, including nitric-oxide (NO)-mediated potassium (K+) efflux at the tips of roots and accumulation of reactive oxygen species (ROS) and reactive nitrogen (RNS) in roots, resulting in physiological stress. We focus on the root system, as the first point of contact with Fe in soil, and describe the key processes engaged in Fe transport, distribution, binding, and other mechanisms that are drawn upon to defend against high-Fe stress. We describe the root-system regulation of key physiological processes and of morphological development through signaling substances such as ethylene, auxin, reactive oxygen species, and nitric oxide, and discuss gene-expression responses under high Fe. We especially focus on studies on the physiological and molecular mechanisms in rice and Arabidopsis under high Fe, hoping to provide a valuable theoretical basis for improving the ability of crop roots to adapt to soil Fe toxicity.

Novel lignin-based extracellular barrier in glandular trichome.

Successful biochemical reactions in organisms necessitate compartmentalization of the requisite components. Glandular trichomes (GTs) act as compartments for the synthesis and storage of specialized compounds. These compounds not only are crucial for the survival of plants under biotic and abiotic stresses but also have medical and commercial value for humans. However, the mechanisms underlying compartmentalization remain unclear. Here we identified a novel structure that is indispensable for the establishment of compartments in cucumber GTs. Silica, a specialized compound, is deposited on the GTs and is visible on the surface of the fruit as a white powder, known as bloom. This deposition provides resistance against pathogens and prevents water loss from the fruits1. Using the cucumber bloomless mutant2, we discovered that a lignin-based cell wall structure in GTs, named 'neck strip', achieves compartmentalization by acting as an extracellular barrier crucial for the silica polymerization. This structure is present in the GTs of diverse plant species. Our findings will enhance the understanding of the biosynthesis of unique compounds in trichomes and provide a basis for improving the production of compounds beneficial to humans.

CsHLS1-CsSCL28 module regulates compact plant architecture in cucumber.

Increased planting densities boost crop yields. A compact plant architecture facilitates dense planting. However, the mechanisms regulating compact plant architecture in cucurbits remain unclear. In this study, we identified a cucumber (Cucumis sativus) compact plant architecture (cpa1) mutant from an ethyl methane sulfonate (EMS)-mutagenized library that exhibited distinctive phenotypic traits, including reduced leaf petiole angle and leaf size. The candidate mutation causes a premature stop codon in CsaV3_1G036420, which shares similarity to Arabidopsis HOOKLESS 1 (HLS1) encoding putative histone N-acetyltransferase (HAT) protein and was named CsHLS1. Consistent with the mutant phenotype, CsHLS1 was predominantly expressed in leaf petiole bases and leaves. Constitutive overexpressing CsHLS1 in cpa1 restored the wild-type plant architecture. Knockout of CsHLS1 resulted in reduces leaf petiole angle and leaf size and as well as decreased acetylation levels. Furthermore, CsHLS1 directly interacted with CsSCL28 and negatively regulated compact plant architecture in cucumber. Importantly, CsHLS1 knockout increased the photosynthesis rate and leaf nitrogen in cucumbers, thereby maintaining cucumber yield at normal density. Overall, our research provides valuable genetic breeding resource and gene target for creating a compact plant architecture for dense cucumber planting.

A lignin-derived material improves plant nutrient bioavailability and growth through its metal chelating capacity.

The lignocellulosic biorefinery industry can be an important contributor to achieving global carbon net zero goals. However, low valorization of the waste lignin severely limits the sustainability of biorefineries. Using a hydrothermal reaction, we have converted sulfuric acid lignin (SAL) into a water-soluble hydrothermal SAL (HSAL). Here, we show the improvement of HSAL on plant nutrient bioavailability and growth through its metal chelating capacity. We characterize HSAL's high ratio of phenolic hydroxyl groups to methoxy groups and its capacity to chelate metal ions. Application of HSAL significantly promotes root length and plant growth of both monocot and dicot plant species due to improving nutrient bioavailability. The HSAL-mediated increase in iron bioavailability is comparable to the well-known metal chelator ethylenediaminetetraacetic acid. Therefore, HSAL promises to be a sustainable nutrient chelator to provide an attractive avenue for sustainable utilization of the waste lignin from the biorefinery industry.

CASP microdomain formation requires cross cell wall stabilization of domains and non-cell autonomous action of LOTR1.

Efficient uptake of nutrients in both animal and plant cells requires tissue-spanning diffusion barriers separating inner tissues from the outer lumen/soil. However, we poorly understand how such contiguous three-dimensional superstructures are formed in plants. Here, we show that correct establishment of the plant Casparian Strip (CS) network relies on local neighbor communication. We show that positioning of Casparian Strip membrane domains (CSDs) is tightly coordinated between neighbors in wild-type and that restriction of domain formation involves the putative extracellular protease LOTR1. Impaired domain restriction in lotr1 leads to fully functional CSDs at ectopic positions, forming 'half strips'. LOTR1 action in the endodermis requires its expression in the stele. LOTR1 endodermal expression cannot complement, while cortex expression causes a dominant-negative phenotype. Our findings establish LOTR1 as a crucial player in CSD positioning acting in a directional, non-cell-autonomous manner to restrict and coordinate CS positioning.

Molecular functions of nitric oxide and its potential applications in horticultural crops.

Nitric oxide (NO) regulates plant growth, enhances nutrient uptake, and activates disease and stress tolerance mechanisms in most plants, making NO a potential tool for use in improving the yield and quality of horticultural crop species. Although the use of NO in horticulture is still in its infancy, research on NO in model plant species has provided an abundance of valuable information on horticultural crop species. Emerging evidence implies that the bioactivity of NO can occur through many potential mechanisms but occurs mainly through S-nitrosation, the covalent and reversible attachment of NO to cysteine thiol. In this context, NO signaling specifically affects crop development, immunity, and environmental interactions. Moreover, NO can act as a fumigant against a wide range of postharvest diseases and pests. However, for effective use of NO in horticulture, both understanding and exploring the biological significance and potential mechanisms of NO in horticultural crop species are critical. This review provides a picture of our current understanding of how NO is synthesized and transduced in plants, and particular attention is given to the significance of NO in breaking seed dormancy, balancing root growth and development, enhancing nutrient acquisition, mediating stress responses, and guaranteeing food safety for horticultural production.

Induction of S-nitrosoglutathione reductase protects root growth from ammonium toxicity by regulating potassium homeostasis in Arabidopsis and rice.

Ammonium (NH4+) is toxic to root growth in most plants already at moderate levels of supply, but mechanisms of root growth tolerance to NH4+ remain poorly understood. Here, we report that high levels of NH4+ induce nitric oxide (NO) accumulation, while inhibiting potassium (K+) acquisition via SNO1 (sensitive to nitric oxide 1)/SOS4 (salt overly sensitive 4), leading to the arrest of primary root growth. High levels of NH4+ also stimulated the accumulation of GSNOR (S-nitrosoglutathione reductase) in roots. GSNOR overexpression improved root tolerance to NH4+. Loss of GSNOR further induced NO accumulation, increased SNO1/SOS4 activity, and reduced K+ levels in root tissue, enhancing root growth sensitivity to NH4+. Moreover, the GSNOR-like gene, OsGSNOR, is also required for NH4+ tolerance in rice. Immunoblotting showed that the NH4+-induced GSNOR protein accumulation was abolished in the VTC1- (vitamin C1) defective mutant vtc1-1, which is hypersensititive to NH4+ toxicity. GSNOR overexpression enhanced vtc1-1 root tolerance to NH4+. Our findings suggest that induction of GSNOR increases NH4+ tolerance in Arabidopsis roots by counteracting NO-mediated suppression of tissue K+, which depends on VTC1 function.

The Emerging Role of GSNOR in Oxidative Stress Regulation.

Oxidative stress is a common event in aerobic organisms and a fundamental and unavoidable cost of the aerobic lifestyle. Reactive oxygen and nitrogen species (ROS/RNS) and iron (Fe) are the most common agents that trigger oxidative stress. A conserved enzyme in the S-nitrosoglutathione (GSNO) metabolism, GSNO reductase (GSNOR), modulates a multitude of abiotic and biotic stress responses. In this review, we focus on the emerging role of GSNOR as a master regulator in oxidative stress through its regulation of the interaction of ROS, RNS, and Fe, and highlight recent discoveries in post-translational modifications of GSNOR and functional variations of natural GSNOR variants during oxidative stress. Recent advances in understanding GSNOR regulation show promise for the modulation of oxidative stress in plants.

Melatonin: A master regulator of plant development and stress responses.

Melatonin is a pleiotropic molecule with multiple functions in plants. Since the discovery of melatonin in plants, numerous studies have provided insight into the biosynthesis, catabolism, and physiological and biochemical functions of this important molecule. Here, we describe the biosynthesis of melatonin from tryptophan, as well as its various degradation pathways in plants. The identification of a putative melatonin receptor in plants has led to the hypothesis that melatonin is a hormone involved in regulating plant growth, aerial organ development, root morphology, and the floral transition. The universal antioxidant activity of melatonin and its role in preserving chlorophyll might explain its anti-senescence capacity in aging leaves. An impressive amount of research has focused on the role of melatonin in modulating postharvest fruit ripening by regulating the expression of ethylene-related genes. Recent evidence also indicated that melatonin functions in the plant's response to biotic stress, cooperating with other phytohormones and well-known molecules such as reactive oxygen species and nitric oxide. Finally, great progress has been made towards understanding how melatonin alleviates the effects of various abiotic stresses, including salt, drought, extreme temperature, and heavy metal stress. Given its diverse roles, we propose that melatonin is a master regulator in plants.

An SMU Splicing Factor Complex Within Nuclear Speckles Contributes to Magnesium Homeostasis in Arabidopsis.

Mg2+ is among the most abundant divalent cations in living cells. In plants, investigations on magnesium (Mg) homeostasis are restricted to the functional characterization of Mg2+ transporters. Here, we demonstrate that the splicing factors SUPPRESSORS OF MEC-8 AND UNC-52 1 (SMU1) and SMU2 mediate Mg homeostasis in Arabidopsis (Arabidopsis thaliana). A low-Mg sensitive Arabidopsis mutant was isolated, and the causal gene was identified as SMU1 Disruption of SMU2, a protein that can form a complex with SMU1, resulted in a similar low-Mg sensitive phenotype. In both mutants, an Mg2+ transporter gene, Mitochondrial RNA Splicing 2 (MRS2-7), showed altered splicing patterns. Genetic evidence indicated that MRS2-7 functions in the same pathway as SMU1 and SMU2 for low-Mg adaptation. In contrast with previous results showing that the SMU1-SMU2 complex is the active form in RNA splicing, MRS2-7 splicing was promoted in the smu2 mutant overexpressing SMU1, indicating that complex formation is not a prerequisite for the splicing. We found here that formation of the SMU1-SMU2 complex is an essential step for their compartmentation in the nuclear speckles, a type of nuclear body enriched with proteins that participate in various aspects of RNA metabolism. Taken together, our study reveals the involvement of the SMU splicing factors in plant Mg homeostasis and provides evidence that complex formation is required for their intranuclear compartmentation.

Circulating Exosomal Gastric Cancer-Associated Long Noncoding RNA1 as a Biomarker for Early Detection and Monitoring Progression of Gastric Cancer: A Multiphase Study.

Importance: The gastric cancer (GC)-associated long noncoding RNA1 (lncRNA-GC1) plays an important role in gastric carcinogenesis. However, exosomal lncRNA-GC1 and its potential role in GC are poorly understood. Objective: To evaluate the diagnostic value of circulating exosomal lncRNA-GC1 for early detection and monitoring progression of GC. Design, Setting, and Participants: We performed a multiphase investigation of circulating exosomal lncRNA-GC1 for early detection of GC involving consecutive patients with GC (n = 522), patients with gastric precancerous lesions (n = 85), and healthy donor individuals (HDs; n = 219) from December 2016 to February 2019 at Chinese People's Liberation Army General Hospital, China. LncRNA-GC1 was measured by reverse transcription-polymerase chain reaction by independent researchers who had no access to patients' information. Receiver operating characteristic curves were used to calculate diagnostic efficiency in comparison between lncRNA-GC1 and 3 traditional biomarkers (carcinoembryonic antigen [CEA], cancer antigen 72-4 [CA72-4], and CA19-9). Main Outcomes and Measures: Assessment of diagnostic efficiency on the basis of area under curve (AUC), specificity, and sensitivity. Results: Of the 826 patients included in the study, 508 were men (61.5%), and the median age of all patients was 60 years (range, 28-82 years). In the test phase, lncRNA-GC1 achieved better diagnostic performance than the standard biomarkers CEA, CA72-4, and CA19-9 (AUC = 0.9033) for distinguishing between the patients with GC and HDs. Additionally, exosomal lncRNA-GC1 levels were significantly higher in culture media from GC cells compared with those of normal gastric epithelial cells (t = 5.310; P = .002). In the verification phase, lncRNA-GC1 retained its diagnostic efficiency in discriminating patients with GC from those with gastric precancerous lesions as well from HDs. Moreover, lncRNA-GC1 exhibited a higher AUC compared with those of CEA, CA72-4, and CA19-9 for early detection of GC with sufficient specificity and sensitivity, especially for patients with GC with negative standard biomarkers. Moreover, the levels of circulating exosomal lncRNA-GC1 were significantly associated with GC from early to advanced stages (HD vs stage I, t = 20.98; P < .001; stage I vs stage II, t = 2.787; P = .006; stage II vs stage III, t = 4.471; P < .001; stage III vs stage IV, t = 1.023; P = .30), independent of pathological grading and Lauren classification (pathological grading: HD vs G1, t = 21.09; P < .001; G1 vs G2, t = 0.3718; P = .71; G2 vs G3, t = 0.3598; P = .72; Lauren classification: t = 24.81; P <.001). In the supplemental phase, the levels of circulating exosomal lncRNA-GC1 were consistent with those in GC tissues and cells and were higher compared with those in normal tissues and cells. Furthermore, the levels of circulating lncRNA-GC1 were unchanged after exosomes were treated with RNase and remained constant after prolonged exposure to room temperature or after repeated freezing and thawing (t = 1.443; P = .39). Total circulating lncRNA-GC1 was nearly all packaged within exosomes rather than a free form in plasma. Conclusions and Relevence: Circulating exosomal lncRNA-GC1 may serve as a noninvasive biomarker for detecting early-stage GC and for monitoring disease progression. Combining circulating exosomal lncRNA-GC1 detection with endoscopy could improve the early diagnostic rate of GC.

Enhancement of polyphenolic metabolism as an adaptive response of lettuce (Lactuca sativa) roots to aluminum stress.

Polyphenols, pivotal secondary metabolites, are involved in plant adaption to abiotic stresses. Here, we investigated the role and metabolism profile of polyphenols under aluminum (Al) stress in different lettuce genotypes grown in 0.5 mM CaCl2 solution with AlCl3 (pH = 4.5). The complementary use of high-resolution mass spectrometry and quantitative biochemical approaches allowed the characterization of total and unique phenols, as well as their roles in Al tolerance. By comparing the most tolerant and sensitive genotype, 8 polyphenols, including 4 phenolic acids, 2 flavonoids, 1 xanthone and 1 unknown compound, were identified in the roots of the tolerant genotype. The total phenolic and flavonoid contents significantly increased in the tolerant genotype under Al stress. Seedlings with more phenolic accumulation usually performed greater Al tolerance. Meanwhile, principal enzymes related to phenolic biosynthesis significantly increased in roots of the tolerance genotype after Al treatment, with phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase, and 4-coumarate coenzyme A ligase increased by 16, 18 and 30%, respectively. The elevated total phenolics were significantly suppressed by AIP, a highly specific PAL inhibitor. Consequently, the antioxidant capacity was inhibited, leading to lettuce sensitivity to Al stress. These results clearly suggested the enhancement of unique polyphenolic biosynthesis as an adaptive strategy of lettuce to Al stress by protecting plants from oxidative stress.

Callose Synthesis Suppresses Cell Death Induced by Low-Calcium Conditions in Leaves.

Despite the importance of preventing calcium (Ca) deficiency disorders in agriculture, knowledge of the molecular mechanisms underlying plant adaptations to low-Ca conditions is limited. In this study, we provide evidence for a crucial involvement of callose synthesis in the survival of Arabidopsis (Arabidopsis thaliana) under low-Ca conditions. A mutant sensitive to low-Ca conditions, low calcium sensitive3 (lcs3), exhibited high levels of cell death in emerging leaves and had defects in its expanding true leaves under low-Ca conditions. Further analyses showed that the causal mutation was located in a putative ß-1,3-glucan (callose) synthase gene, GLUCAN SYNTHASE-LIKE10 (GSL10). Yeast complementation assay results showed that GSL10 encodes a functional callose synthase. Ectopic callose significantly accumulated in wild-type plants under low-Ca conditions, but at a low level in lcs3 The low-Ca sensitivity of lcs3 was phenocopied by the application of callose synthase inhibitors in wild-type plants, which resulted in leaf expansion failure, cell death, and reduced ectopic callose levels under low-Ca conditions. Transcriptome analyses showed that the expression of genes related to cell wall and defense responses was altered in both wild-type plants under low-Ca conditions and in lcs3 under normal-Ca conditions, suggesting that GSL10 is required for the alleviation of both cell wall damage and defense responses caused by low Ca levels. These results suggest that callose synthesis is essential for the prevention of cell death under low-Ca conditions and plays a key role in plants' survival strategies under low-Ca conditions.

GSNOR provides plant tolerance to iron toxicity via preventing iron-dependent nitrosative and oxidative cytotoxicity.

Iron (Fe) is essential for life, but in excess can cause oxidative cytotoxicity through the generation of Fe-catalyzed reactive oxygen species. It is yet unknown which genes and mechanisms can provide Fe-toxicity tolerance. Here, we identify S-nitrosoglutathione-reductase (GSNOR) variants underlying a major quantitative locus for root tolerance to Fe-toxicity in Arabidopsis using genome-wide association studies and allelic complementation. These variants act largely through transcript level regulation. We further show that the elevated nitric oxide is essential for Fe-dependent redox toxicity. GSNOR maintains root meristem activity and prevents cell death via inhibiting Fe-dependent nitrosative and oxidative cytotoxicity. GSNOR is also required for root tolerance to Fe-toxicity throughout higher plants such as legumes and monocots, which exposes an opportunity to address crop production under high-Fe conditions using natural GSNOR variants. Overall, this study shows that genetic or chemical modulation of the nitric oxide pathway can broadly modify Fe-toxicity tolerance.

Cloning, expression profiling, and effects of fasting status on neuropeptide Y in Schizothorax davidi.

To better comprehend the mechanism that neuropeptide Y (npy) regulates feeding in Schizothorax davidi, we cloned and identified the full-length cDNA sequence of the npy gene in this species using RACE technology. Subsequently, we explored the npy mRNA distribution in 18 tissues and investigated the expression of npy mRNA at postprandial and fasting stages. We found that the npy full-length cDNA sequence is 803 bp. Moreover, npy mRNAs extensively expressed in all detected tissues, with the highest expression in hypothalamus. In postprandial study, the expression of npy mRNA in the hypothalamus was significantly decreased after eating (p < 0.01). In addition, the expression of the npy gene was significantly increased on the fifth day after fasting (p < 0.05). However, after refeeding, the expression of the npy gene was decreased significantly on days 9, 11, and 14 (p < 0.01). Our research suggest that npy may have an orexigenic role in S. davidi. PRACTICAL APPLICATIONS: S. davidi, a coldwater fish native to China, has high economic value, and it has gained great popularity. To date, there is still no large-scale breeding of S. davidi in China. How to strengthen the production performance of S. davidi is a hot research area. Neuropeptide Y (NPY), a 36-amino-acid single-chain polypeptide, is one of the main appetite regulation factors. However, to date, no studies have reported on the biological function of npy in the feeding of S. davidi. In our study, we revealed that the trend of hypothalamic npy expression during the postprandial and fasting stages. The results suggested that npy might be an appetite-promoting factor in this species. Overall, we provide the theoretical basis for how to strengthen the production performance of S. davidi through appetite regulation.

Three forms of cocaine- and amphetamine-regulated transcript may be involved in food intake regulation in gibel carp (Carassius auratus gibelio).

In fish, as in mammals, several studies have demonstrated that the cocaine- and amphetamine-regulated transcript (CART) plays an important role in feeding. However, thus far, the function of CART in gibel carp (Carassius auratus gibelio) feeding regulation has not been reported. In our study, we first identified three forms of CART peptide precursors from gibel carp brain and named these CART-1, CART-2, and CART-3. The full-length cDNA sequences of CART-1, CART-2, and CART-3 were 616 bp, 705 bp, and 760 bp, respectively, encoding peptides of 118, 120, and 104 amino acid residues. We detected mRNA expression of CART-1, CART-2, and CART-3 in a wide range of peripheral and central tissues, with the highest expression detected in the brain. After a meal, mRNA expression of CART-1, CART-2, and CART-3 was significantly elevated, suggesting that CART-1, CART-2, and CART-3 may act as postprandial satiety signals. Moreover, mRNA expression of all three CART-1, CART-2, and CART-3 was significantly reduced during fasting and significantly elevated with refeeding. Our findings indicate that CART-1, CART-2, and CART-3 might function as a satiety factor in the gibel carp.

Structural and functional characterization of peptide YY on feeding in Schizothorax davidi.

Several studies have demonstrated that the neuropeptide peptide YY (PYY) plays an important role in feeding in mammals and fish. However, thus far, the feeding regulation function of PYY in Schizothorax davidi has not been well understood. Here, we identified the full-length cDNA sequence of PYY in S. davidi for the first time. S. davidi PYY contains 803 bp nucleotides including a 328 bp 3' untranslated region (UTR), a 181 bp 5' UTR, and a 294 bp open reading frame encoding a peptide of 97 amino acids. S. davidi PYY expression was observed in almost all tissues, with the highest expression detected in the hypothalamus. PYY mRNA expression in the hypothalamus was significantly elevated after a meal (P < 0.01), and significantly decreased after fasting (P < 0.01). PYY expression levels were increased sharply following refeeding after 9 days (P < 0.01), suggesting that it might function as a satiety factor in S. davidi.

Characterization of the complete mitochondrial genome of the endangered species Schizothorax integrilabiatus.

Schizothorax integrilabiatus is an endangered fish species found in the Buqun Lake of Qinghai-Tibet Plateau. In this study, we determined the complete mitochondrial genome sequence of the S. integrilabiatus. The circular mitochondrial genome was 16,621 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a control region (D-loop). The overall base composition is A 30.1%, C 26.9%, G 17.4%, and T 25.6%, with a high A + T content (55.7%). Further, phylogenetic analysis suggested that S. integrilabiatus is closely related to species of S. plagiostomus, and then clustered into a clade with other Schizothoracinae species. This work provides additional molecular information for studying S. integrilabiatus conservation genetics and evolutionary relationships.

Role of LOTR1 in Nutrient Transport through Organization of Spatial Distribution of Root Endodermal Barriers.

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