Early differentiation of Casparian strip mediated by nitric oxide is required for efficient K transport under low K conditions in Arabidopsis.

The Casparian strip (CS) is a cell wall modification made of lignin that functions as an apoplastic barrier in the root endodermis to restrict nutrient and water transport between the soil and stele. CS formation is affected by nutritional conditions, and its physiological roles have been discussed. This study found that low K condition affects CS permeability, lignin deposition, and MYB36 mRNA accumulation. To understand the mechanism underlying these findings, we focused on nitric oxide (NO). NO is known to act as a signaling molecule and participates in cell wall synthesis, especially for lignin composition. However, the mechanism by which NO affects lignin deposition and corrects CS formation in the plant roots remains unclear. Through combining fluorescent observation with histological stains, we demonstrated that the root endodermal cell lignification response to low-potassium (K) conditions is mediated by NO through the MYB36-associated lignin-polymerizing pathway. Furthermore, we discovered the noteworthy ability of NO to maintain nutrient homeostasis for adaptation to low K conditions by affecting the correct apoplastic barrier formation of CS. Collectively, our results suggest that NO is required for the lignification and apoplastic barrier formation in the root endodermis during adaptation to low K conditions, which revealing the novel physiological roles of CS under low nutrient conditions and making a significant contribution to CS biology.

A pearl millet plasma membrane protein, PgPM19, facilitates seed germination through the negative regulation of abscisic acid-associated genes under salinity stress in Arabidopsis thaliana.

MAIN CONCLUSION: The pearl millet gene PgPM19 inhibits seed dormancy by negatively regulating the ABA biosynthesis and ABA signaling pathways in response to salinity stress in Arabidopsis. Abscisic acid (ABA) plays a pivotal role in orchestrating plant stress responses and development. However, how the ABA signal is transmitted in response to stresses remains primarily uncertain, particularly in monocotyledonous plants. In this study, PgPM19, a gene whose expression is induced by drought, salinity, heat, and ABA in both leaf and root tissues, was isolated from pearl millet. The expression of PgPM19 in yeast cells did not influence their growth when subjected to mannitol, sorbitol, or NaCl stress. However, Arabidopsis plants overexpressing PgPM19 (PgPM19_OE plants) exhibited increased germination rates, greater fresh weights and longer roots under salinity stress during germination, compared to wild-type (WT) plants. Conversely, the pm19L1 (SALK_075435) mutant, featuring a transfer DNA insertion in a closely related PgPM19 homolog (AT1G04560) in Arabidopsis, demonstrated reduced germination rates and smaller fresh weights under salinity-stressed condition than did WT and PgPM19_OE plants. A pivotal ABA biosynthesis gene, NCED3, ABA signaling pathway genes, such as PYL6 and SnRK2.7, alongside downstream ABI genes and stress-responsive genes RAB28 and RD29, were downregulated in PgPM19_OE plants, as evidenced by both transcriptome analysis and quantitative reverse transcription-PCR. These findings raise the possibility that PgPM19 is involved in regulating seed germination by mediating ABA biosynthesis and signaling pathway in response to salinity stress in Arabidopsis. This study contributes to a better understanding of PgPM19 in response to salinity stress and establishes a foundation for unraveling the crosstalk of stress responses and ABA in Arabidopsis and other plant species.

Arabidopsis KNS3 and its two homologs mediate endoplasmic reticulum-to-plasma membrane traffic of boric acid channels.

Membrane proteins targeted to the plasma membrane are first transported from the endoplasmic reticulum (ER) to the Golgi apparatus. This study explored the mechanisms controlling plasma membrane trafficking of the boric acid channel AtNIP5;1 from the ER. Imaging-based screening using transgenic Arabidopsis identified six mutants in which GFP-NIP5;1 was localized in the ER in addition to the plasma membrane. Genetic mapping and whole-genome resequencing identified the responsible gene in four among the six mutants as KAONASHI3 (KNS3)/SPOTTY1/IMPERFECTIVE EXINE FORMATION. Among the plasma membrane-localized proteins tested, NIP5;1 and its homolog NIP6;1 were retained in the ER of the kns3 mutants. Our genetic analysis further discovered that two homologs of KNS3, KNSTH1 and KNSTH2, were also involved in the ER exit of NIP5;1. In Arabidopsis protoplasts and tobacco leaves, mCherry-fused KNS3 localized to the ER and Golgi, whereas KNSTH2 localized to the ER. The cytosolic C-terminal tail of KNS3 contains amino acids important for Golgi-to-ER trafficking. Furthermore, the ER-to-Golgi trafficking of KNS3 depended on KNSTH1 and KNSTH2, and the accumulation of these three proteins in Arabidopsis roots depended on each other. We propose that KNS3, KNSTH1, and KNSTH2 function as a cargo-receptor complex mediating the ER exit of NIP5;1.

Inhibition of NPR1 Leads to Shoot Growth Improvement under Low-Calcium Conditions in Arabidopsis.

Under low-Ca conditions, plants accumulate salicylic acid (SA) and induce SA-responsive genes. However, the relationship between SA and low-Ca tolerance remains unclear. Here, we demonstrated that the inhibition or suppression of nonexpressor of pathogenesis-related 1 (NPR1) activity, a major regulator of the SA signaling pathway in the defense response, improves shoot growth under low-Ca conditions. Furthermore, mutations in phytoalexin-deficient 4 (PAD4) or enhanced disease susceptibility 1 (EDS1), which are upstream regulators of NPR1, improved shoot growth under low-Ca conditions, suggesting that NPR1 suppressed growth under low-Ca conditions. In contrast, growth of SA induction-deficient 2-2 (sid2-2), which is an SA-deficient mutant, was sensitive to low Ca levels, suggesting that SA accumulation by SID2 was not related to growth inhibition under low-Ca conditions. Additionally, npr1-1 showed low-Ca tolerance, and the application of tenoxicam-an inhibitor of the NPR1-mediated activation of gene expression-also improved shoot growth under low Ca conditions. The low-Ca tolerance of double mutants pad4-1, npr1-1 and eds1-22 npr1-1 was similar to that of the single mutants, suggesting that PAD4 and EDS1 are involved in the same genetic pathway in suppressing growth under low-Ca conditions as NPR1. Cell death and low-Ca tolerance did not correlate among the mutants, suggesting that growth improvement in the mutants was not due to cell death inhibition. In conclusion, we revealed that NPR1 suppresses plant growth under low-Ca conditions and that the other SA-related genes influence plant growth and cell death.

Regulation of ammonium acquisition and use in Oryza longistaminata ramets under nitrogen source heterogeneity.

Oryza longistaminata, a wild rice, vegetatively reproduces and forms a networked clonal colony consisting of ramets connected by rhizomes. Although water, nutrients, and other molecules can be transferred between ramets via the rhizomes, inter-ramet communication in response to spatially heterogeneous nitrogen availability is not well understood. We studied the response of ramet pairs to heterogeneous nitrogen availability using a split hydroponic system that allowed each ramet root to be exposed to different conditions. Ammonium uptake was compensatively enhanced in the sufficient-side root when roots of the ramet pairs were exposed to ammonium-sufficient and ammonium-deficient conditions. Comparative transcriptome analysis revealed that a gene regulatory network for effective ammonium assimilation and amino acid biosynthesis was activated in the sufficient-side roots. Allocation of absorbed nitrogen from the nitrogen-sufficient to the nitrogen-deficient ramets was rather limited. Nitrogen was preferentially used for newly growing axillary buds on the sufficient-side ramets. Biosynthesis of trans-zeatin (tZ), a cytokinin, was upregulated in response to the nitrogen supply, but tZ appeared not to target the compensatory regulation. Our results also implied that the O. longistaminata putative ortholog of rice (Oryza sativa) C-terminally encoded peptide1 plays a role as a nitrogen-deficient signal in inter-ramet communication, providing compensatory upregulation of nitrogen assimilatory genes. These results provide insights into the molecular basis for efficient growth strategies of asexually proliferating plants growing in areas where the distribution of ammonium ions is spatially heterogeneous.

Arabidopsis Glucan Synthase-Like1 (GSL1) Is Required for Tolerance to Low-Calcium Conditions and Exhibits a Function Comparable to GSL10.

Crops that exhibit symptoms of calcium (Ca) deficiency constitute a major agricultural problem. Molecular breeding of resistant cultivars is a promising method for overcoming this problem. However, the involved genes must first be identified. Here, we show that the glucan synthase-like (GSL) 1 gene is essential for low-Ca tolerance in Arabidopsis thaliana. GSL1 is homologous to GSL10, which we previously showed was essential for low-Ca tolerance. Under low-Ca conditions, gsl1 mutants exhibit reduced growth and the onset of necrosis in new leaves. These symptoms are typical of Ca-deficient crops. A grafting experiment suggested that the shoot genotype, but not the root genotype, was important for the suppression of shoot necrosis. The ectopic accumulation of callose under low-Ca conditions was significantly reduced in gsl1 mutants compared with wild-type plants. Because the corresponding single-mutant phenotypes are similar, we investigated the interaction between GSL1 and GSL10 by testing the gsl1 gsl10 double mutant for sensitivity to low-Ca conditions. The double mutant exhibited a more severe phenotype than did the single mutants, indicating that the effects of GSL1 and GSL10 on low-Ca tolerance are additive. Because GSL genes are highly conserved within the plant kingdom, the GSL loci may be useful for breeding low-Ca tolerant crops.

Sorghum Ionomics Reveals the Functional SbHMA3a Allele that Limits Excess Cadmium Accumulation in Grains.

Understanding uptake and redistribution of essential minerals or sequestering of toxic elements is important for optimized crop production. Although the mechanisms controlling mineral transport have been elucidated in rice and other species, little is understood in sorghum-an important C4 cereal crop. Here, we assessed the genetic factors that govern grain ionome profiles in sorghum using recombinant inbred lines (RILs) derived from a cross between BTx623 and NOG (Takakibi). Pairwise correlation and clustering analysis of 22 elements, measured in sorghum grains harvested under greenhouse conditions, indicated that the parental lines, as well as the RILs, show different ionomes. In particular, BTx623 accumulated significantly higher levels of cadmium (Cd) than NOG, because of differential root-to-shoot translocation factors between the two lines. Quantitative trait locus (QTL) analysis revealed a prominent QTL for grain Cd concentration on chromosome 2. Detailed analysis identified SbHMA3a, encoding a P1B-type ATPase heavy metal transporter, as responsible for low Cd accumulation in grains; the NOG allele encoded a functional HMA3 transporter (SbHMA3a-NOG) whose Cd-transporting activity was confirmed by heterologous expression in yeast. BTx623 possessed a truncated, loss-of-function SbHMA3a allele. The functionality of SbHMA3a in NOG was confirmed by Cd concentrations of F2 grains derived from the reciprocal cross, in which the NOG allele behaved in a dominant manner. We concluded that SbHMA3a-NOG is a Cd transporter that sequesters excess Cd in root tissues, as shown in other HMA3s. Our findings will facilitate the isolation of breeding cultivars with low Cd in grains or in exploiting high-Cd cultivars for phytoremediation.

Systemic Regulation of Iron Acquisition by Arabidopsis in Environments with Heterogeneous Iron Distributions.

Nutrient distribution within the soil is generally heterogeneous. Plants, therefore, have evolved sophisticated systemic processes enabling them to optimize their nutrient acquisition efficiency. By organ-to-organ communication in Arabidopsis thaliana, for instance, iron (Fe) starvation in one part of a root drives the upregulation of a high-affinity Fe-uptake system in other root regions surrounded by sufficient levels of Fe. This compensatory response through Fe-starvation-triggered organ-to-organ communication includes the upregulation of Iron-regulated transporter 1 (IRT1) gene expression on the Fe-sufficient side of the root; however, the molecular basis underlying this long-distance signaling remains unclear. Here, we analyzed gene expression by RNA-seq analysis of Fe-starved split-root cultures. Genome-wide expression analysis showed that localized Fe depletion in roots upregulated several genes involved in Fe uptake and signaling, such as IRT1, in a distant part of the root exposed to Fe-sufficient conditions. This result indicates that long-distance signaling for Fe demand alters the expression of a subset of genes responsible for Fe uptake and coumarin biosynthesis to maintain a level of Fe acquisition sufficient for the entire plant. Loss of IRON MAN/FE-UPTAKE-INDUCING PEPTIDE (IMA/FEP) leads to the disruption of compensatory upregulation of IRT1 in the root surrounded by sufficient Fe. In addition, our split-root culture-based analysis provides evidence that the IMA3/FEP1-MYB10/72 pathway mediates long-distance signaling in Fe homeostasis through the regulation of coumarin biosynthesis. These data suggest that the signaling of IMA/FEP, a ubiquitous family of metal-binding peptides, is critical for organ-to-organ communication in response to Fe starvation under heterogeneous Fe conditions in the surrounding environment.

Root-specific activation of plasma membrane H+-ATPase 1 enhances plant growth and shoot accumulation of nutrient elements under nutrient-poor conditions in Arabidopsis thaliana.

Plasma membrane (PM) H+-ATPase contributes to nutrient uptake and stomatal opening by creating proton gradient across the membrane. Previous studies report that a dominant mutation in the OPEN STOMATA2 locus (OST2-2D) constitutively activates Arabidopsis PM H+-ATPase 1 (AHA1), which enlarges proton motive force for root nutrient uptake. However, the stomatal opening is also constitutively enhanced in the ost2-2D, causing drought hypersensitivity. To develop plants with improved nutrient acquisition and normal stomatal movement, we generated grafted plants (scion/rootstock: Col-0 (WT)/ost2-2D), and compared their growth, nutrient element content, and transcriptomes with those of control plants (WT/WT) under nutrient-rich or nutrient-poor conditions. WT/ost2-2D shoots had larger weights, rosette diameter, leaf blade area, and content of C, N, K, Ca, S, P, Mg, Na, Mn, B, Co, and Mo under nutrient-poor conditions compared with WT/WT shoots. The root weights and primary root length were greater in WT/ost2-2D plants than in WT/WT plants under both nutrient conditions. Root expression of the high-affinity nitrate transporter NRT2.1, potassium transporter HAK5, and divalent cation transporter IRT1 was higher in WT/ost2-2D plants than in WT/WT plants under nutrient-poor conditions. These results suggest that root-specific activation of PM H+-ATPase enhances plant growth by increasing root uptake of nutrient elements under nutrient-poor conditions. Our study presents a novel approach to improving nutrient uptake efficiency in crops for low-input sustainable agriculture.

Tonoplast-Localized OsMOT1;2 Participates in Interorgan Molybdate Distribution in Rice.

Molybdenum (Mo) is an essential element for plant growth and is utilized by several key enzymes in biological redox processes. Rice assimilates molybdate ions via OsMOT1;1, a transporter with a high affinity for molybdate. However, other systems involved in the molecular transport of molybdate in rice remain unclear. Here, we characterized OsMOT1;2, which shares amino acid sequence similarity with AtMOT1;2 and functions in vacuolar molybdate export. We isolated a rice mutant harboring a complete deletion of OsMOT1;2. This mutant exhibited a significantly lower grain Mo concentration than the wild type (WT), but its growth was not inhibited. The Mo concentration in grains was restored by the introduction of WT OsMOT1;2. The OsMOT1;2-GFP protein was localized to the vacuolar membrane when transiently expressed in rice protoplasts. At the reproductive growth stage of the WT plant, OsMOT1;2 was highly expressed in the 2nd and lower leaf blades and nodes. The deletion of OsMOT1;2 impaired interorgan Mo allocation in aerial parts: relative to the WT, the mutant exhibited decreased Mo levels in the 1st and 2nd leaf blades and grains but increased Mo levels in the 2nd and lower leaf sheaths, nodes and internodes. When the seedlings were exposed to a solution with a high KNO3 concentration in the absence of Mo, the mutant exhibited significantly lower nitrate reductase activity in the shoots than the WT. Our results suggest that OsMOT1;2 plays an essential role in interorgan Mo distribution and molybdoenzyme activity in rice.

Growth and Nitrate Reductase Activity Are Impaired in Rice Osnlp4 Mutants Supplied with Nitrate.

Nitrate is an important nutrient and signaling molecule in plants, which modulates the expression of many genes and regulates plant growth. In paddy-grown rice (Oryza sativa), nitrogen is mostly supplied in the form of ammonium but can also be supplied in the form of nitrate. Several nitrogen transporters and nitrate assimilation enzymes have been identified and functionally characterized in rice. However, little is known regarding the nitrate sensing system in rice, and the regulatory mechanisms of nitrate-related genes remain to be elucidated. In recent years, NIN-like proteins (NLPs) have been described as key transcription factors of nitrogen responses in Arabidopsis thaliana, which implies that OsNLP4 is involved in the regulation of nitrate assimilation and nitrogen use efficiency in rice. Here, we show that OsNLP4 can influence plant growth by affecting nitrate reductase (NR) activity. The growth of OsNLP4 knockdown mutants was reduced when nitrate was supplied, but not when ammonium was supplied. The nitrate concentration was significantly reduced in osnlp4 mutants. Furthermore, the concentrations of iron and molybdenum, essential elements for NR activity, were reduced in OsNLP4 knockdown mutants. We propose that, in addition to the regulation of gene expression within the nitrate signaling pathway, OsNLP4 can affect the NR activity and nitrate-dependent growth of rice. Our results support a working model for the role of OsNLP4 in the nitrate signaling pathway.

A Novel Signaling Pathway Required for Arabidopsis Endodermal Root Organization Shapes the Rhizosphere Microbiome.

The Casparian strip (CS) constitutes a physical diffusion barrier to water and nutrients in plant roots, which is formed by the polar deposition of lignin polymer in the endodermis tissue. The precise pattern of lignin deposition is determined by the scaffolding activity of membrane-bound Casparian Strip domain proteins (CASPs), but little is known of the mechanism(s) directing this process. Here, we demonstrate that Endodermis-specific Receptor-like Kinase 1 (ERK1) and, to a lesser extent, ROP Binding Kinase1 (RBK1) are also involved in regulating CS formation, with the former playing an essential role in lignin deposition as well as in the localization of CASP1. We show that ERK1 is localized to the cytoplasm and nucleus of the endodermis and that together with the circadian clock regulator, Time for Coffee (TIC), forms part of a novel signaling pathway necessary for correct CS organization and suberization of the endodermis, with their single or combined loss of function resulting in altered root microbiome composition. In addition, we found that other mutants displaying defects in suberin deposition at the CS also display altered root exudates and microbiome composition. Thus, our work reveals a complex network of signaling factors operating within the root endodermis that establish both the CS diffusion barrier and influence the microbial composition of the rhizosphere.

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.

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.

Rosmarinic Acid and Sodium Citrate Have a Synergistic Bacteriostatic Effect against Vibrio Species by Inhibiting Iron Uptake.

BACKGROUND: New strategies are needed to combat multidrug-resistant bacteria. The restriction of iron uptake by bacteria is a promising way to inhibit their growth. We aimed to suppress the growth of Vibrio bacterial species by inhibiting their ferric ion-binding protein (FbpA) using food components. METHODS: Twenty spices were selected for the screening of FbpA inhibitors. The candidate was applied to antibacterial tests, and the mechanism was further studied. RESULTS: An active compound, rosmarinic acid (RA), was screened out. RA binds competitively and more tightly than Fe3+ to VmFbpA, the FbpA from V. metschnikovii, with apparent KD values of 8 µM vs. 17 µM. Moreover, RA can inhibit the growth of V. metschnikovii to one-third of the control at 1000 µM. Interestingly, sodium citrate (SC) enhances the growth inhibition effect of RA, although SC only does not inhibit the growth. The combination of RA/SC completely inhibits the growth of not only V. metschnikovii at 100/100 µM but also the vibriosis-causative pathogens V. vulnificus and V. parahaemolyticus, at 100/100 and 1000/100 µM, respectively. However, RA/SC does not affect the growth of Escherichia coli. CONCLUSIONS: RA/SC is a potential bacteriostatic agent against Vibrio species while causing little damage to indigenous gastrointestinal bacteria.

Plasma Membrane-Associated Ca2+-Binding Protein PCaP1 is Involved in Root Hydrotropism of Arabidopsis thaliana.

Root hydrotropism is an essential growth response to water potential gradients in plants. To understand the mechanism, fundamental elements such as MIZU-KUSSEI 1 (MIZ1) have been investigated extensively. We investigated the physiological role of a plasma membrane-associated cation-binding protein (PCaP1) and examined the effect of PCaP1 loss-of-function mutations on root hydrotropism. pcap1 knockout mutants showed a defect in root bending as a hydrotropic response, although gravitropism was normal in pcap1 mutants. When pcap1 seedlings were treated with abscisic acid, a negative regulator of gravitropism, the seedlings showed normal gravitropism. The hydrotropism defect in pcap1 mutants was clearly rescued by introducing the genomic sequence of PCaP1 with an endodermis-specific promoter. Analysis of PCaP1-greenfluorescent protein-expressing roots by confocal laser scanning microscopy revealed that PCaP1 was stably associated with the plasma membrane in most cells, but in the cytoplasm of endodermal cells at the bending region. Furthermore, we prepared a transgenic line overexpressing MIZ1 on the pcap1 background and found that the pcap1 hydrotropism defect was rescued. Our results indicate that PCaP1 in the endodermal cells of the root elongation zone is involved in the hydrotropic response. We suggest that PCaP1 contributes to hydrotropism through a MIZ1-independent pathway or as one of the upstream components that transduce water potential signals to MIZ1.

SQUAMOSA promoter-binding protein-like 7 mediates copper deficiency response in the presence of high nitrogen in Arabidopsis thaliana.

KEY MESSAGE: SQUAMOSA promoter-binding protein-like 7 mediates copper deficiency response in the presence of high nitrogen even with the sufficient level of copper in Arabidopsis thaliana. Under copper (Cu) deficiency, accumulation of mRNA encoding two Cu/Zn superoxide dismutases, CSD1 and CSD2, is downregulated to save Cu for plastocyanin. This downregulation depends on miR398 and is under the control of SQUAMOSA promoter-binding protein-like7 (SPL7). Arabidopsis seedlings are routinely cultured on Murashige and Skoog medium. However, the high nitrogen (N) content of the medium (60 mM) has been shown to induce a similar response to Cu deficiency. The mRNA and protein levels of CSD1 and CSD2 are reduced under high N conditions, even if the Cu concentration in the medium is sufficient (0.1-0.5 µM). In this study, we show that this symptom, similar to the Cu deficiency, occurred in the presence of high N largely depending on SPL7, suggesting that plants actually sensed Cu deficiency. However, a change in N concentration in the medium did not influence the total Cu concentration in shoots or roots. High N did not increase the protein content in leaves but facilitated rapid seedling growth. We speculate that this rapid growth causes a continuous Cu deficiency mainly because of high Cu uptake by mesophyll cells in the leaves. This idea was supported by the observation that plastocyanin did not overaccumulate at the range of 0.1-0.5 µM Cu with 30 mM N. In contrast, in the presence of 5 µM Cu with 30 mM N, plants accumulate more Cu in plastocyanin in the thylakoid lumen, resulting in a slight Cu deficiency in the chloroplast stroma. This process is independent of SPL7.

LjMOT1, a high-affinity molybdate transporter from Lotus japonicus, is essential for molybdate uptake, but not for the delivery to nodules.

Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high-affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate-mutagenized line (ljmot1) decreased by 70-95% compared with wild-type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp292 to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F2 progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60-70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the Km was 182 nm. LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons-removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not for Mo delivery to the nodules.

Screening Arabidopsis thaliana mutants for low sensitivity to manganese identifies novel alleles of NRAMP1 and PGSIP6.

Manganese (Mn) is an essential micronutrient; however, few genes required for growth under low-Mn conditions have been identified. In this study, we isolated Arabidopsis thaliana mutants sensitive to low-Mn conditions from ethyl methanesulfonate-mutagenized seeds. Among them, we identified the causal genes of two mutants. One mutant (35-34) exhibited a short root phenotype and low Mn concentration in the shoots. The other mutant (30-11) exhibited a small shoot phenotype with Mn concentrations similar to the control. Genetic mapping, allelism tests, and gene complementation tests identified the causal genes as At1g80830 (NRAMP1) for 35-34 and At5g18480 (PGSIP6) for 30-11. NRAMP1 was previously reported to be essential for Mn uptake under low-Mn conditions, thus validating our screening method. PGSIP6 encodes inositol phosphorylceramide glucuronosyltransferase, which is involved in glycosyl inositol phosphorylceramide sphingolipid glycosylation. PGSIP6-green fluorescent protein was localized to the Golgi apparatus, which is consistent with its function in the glycosylation of sphingolipids. Our screening identified a novel gene required for low-Mn tolerance, and we also provide new insights towards understanding the physiological function of PGSIP6.

The MYB36 transcription factor orchestrates Casparian strip formation.

The endodermis in roots acts as a selectivity filter for nutrient and water transport essential for growth and development. This selectivity is enabled by the formation of lignin-based Casparian strips. Casparian strip formation is initiated by the localization of the Casparian strip domain proteins (CASPs) in the plasma membrane, at the site where the Casparian strip will form. Localized CASPs recruit Peroxidase 64 (PER64), a Respiratory Burst Oxidase Homolog F, and Enhanced Suberin 1 (ESB1), a dirigent-like protein, to assemble the lignin polymerization machinery. However, the factors that control both expression of the genes encoding this biosynthetic machinery and its localization to the Casparian strip formation site remain unknown. Here, we identify the transcription factor, MYB36, essential for Casparian strip formation. MYB36 directly and positively regulates the expression of the Casparian strip genes CASP1, PER64, and ESB1. Casparian strips are absent in plants lacking a functional MYB36 and are replaced by ectopic lignin-like material in the corners of endodermal cells. The barrier function of Casparian strips in these plants is also disrupted. Significantly, ectopic expression of MYB36 in the cortex is sufficient to reprogram these cells to start expressing CASP1-GFP, correctly localize the CASP1-GFP protein to form a Casparian strip domain, and deposit a Casparian strip-like structure in the cell wall at this location. These results demonstrate that MYB36 is controlling expression of the machinery required to locally polymerize lignin in a fine band in the cell wall for the formation of the Casparian strip.