GmNLP1 and GmNLP4 activate nitrate-induced CLE peptides NIC1a/b to mediate nitrate-regulated root nodulation.

Symbiotic nitrogen fixation is an energy-intensive process, to maintain the balance between growth and nitrogen fixation, high concentrations of nitrate inhibit root nodulation. However, the precise mechanism underlying the nitrate inhibition of nodulation in soybean remains elusive. In this study, CRISPR-Cas9-mediated knockout of GmNLP1 and GmNLP4 unveiled a notable nitrate-tolerant nodulation phenotype. GmNLP1b and GmNLP4a play a significant role in the nitrate-triggered inhibition of nodulation, as the expression of nitrate-responsive genes was largely suppressed in Gmnlp1b and Gmnlp4a mutants. Furthermore, we demonstrated that GmNLP1b and GmNLP4a can bind to the promoters of GmNIC1a and GmNIC1b and activate their expression. Manipulations targeting GmNIC1a and GmNIC1b through knockdown or overexpression strategies resulted in either increased or decreased nodule number in response to nitrate. Additionally, transgenic roots that constitutively express GmNIC1a or GmNIC1b rely on both NARK and hydroxyproline O-arabinosyltransferase RDN1 to prevent the inhibitory effects imposed by nitrate on nodulation. In conclusion, this study highlights the crucial role of the GmNLP1/4-GmNIC1a/b module in mediating high nitrate-induced inhibition of nodulation.

An endophytic fungus interacts with the defensin-like protein OsCAL1 to regulate cadmium allocation in rice.

Defensin-like proteins are conserved in multicellular organisms and contribute to innate immune responses against fungal pathogens. In rice, defensins play a novel role in regulating cadmium (Cd) efflux from the cytosol. However, whether the antifungal activity of defensins correlates with Cd-efflux function remains unknown. In this study, we isolated an endophytic Fusarium, designed Fo10, by a comparative microbiome analysis of rice plants grown in a paddy contaminated with Cd. Fo10 is tolerant to high levels of Cd, but is sensitive to the defensin-like protein OsCAL1, which mediates Cd efflux to the apoplast. We found that Fo10 symbiosis in rice is regulated by OsCAL1 dynamics, and Fo10 coordinates multiple plant processes, including Cd uptake, vacuolar sequestration, efflux to the environment, and formation of Fe plaques in the rhizosphere. These processes are dependent on the salicylic acid signaling pathway to keep Cd levels low in the cytosol of rice cells and to decrease Cd levels in rice grains without any yield penalty. Fo10 also plays a role in Cd tolerance in the poaceous crop maize and wheat, but has no observed effects in the eudicot plants Arabidopsis and tomato. Taken together, these findings provide insights into the mechanistic basis underlying how a fungal endophyte and host plant interact to control Cd accumulation in host plants by adapting defense responses to promote the establishment of a symbiosis that permits adaptation to high-Cd environments.

Dual-function DEFENSIN 8 mediates phloem cadmium unloading and accumulation in rice grains.

Grain cadmium (Cd) is translocated from source to sink tissues exclusively via phloem, though the phloem Cd unloading transporter has not been identified yet. Here, we isolated and functionally characterized a defensin-like gene DEFENSIN 8 (DEF8) highly expressed in rice (Oryza sativa) grains and induced by Cd exposure in seedling roots. Histochemical analysis and subcellular localization detected DEF8 expression preferentially in pericycle cells and phloem of seedling roots, as well as in phloem of grain vasculatures. Further analysis demonstrated that DEF8 is secreted into extracellular spaces possibly by vesicle trafficking. DEF8 bound to Cd in vitro, and Cd efflux from protoplasts as well as loading into xylem vessels decreased in the def8 mutant seedlings compared with the wild type. At maturity, significantly less Cd accumulation was observed in the mutant grains. These results suggest that DEF8 is a dual function protein that facilitates Cd loading into xylem and unloading from phloem, thus mediating Cd translocation from roots to shoots and further allocation to grains, representing a phloem Cd unloading regulator. Moreover, essential mineral nutrient accumulation as well as important agronomic traits were not affected in the def8 mutants, suggesting DEF8 is an ideal target for breeding low grain Cd rice.

The small peptide CEP1 and the NIN-like protein NLP1 regulate NRT2.1 to mediate root nodule formation across nitrate concentrations.

Legumes acquire fixed nitrogen (N) from the soil and through endosymbiotic association with diazotrophic bacteria. However, establishing and maintaining N2-fixing nodules are expensive for the host plant, relative to taking up N from the soil. Therefore, plants suppress symbiosis when N is plentiful and enhance symbiosis when N is sparse. Here, we show that the nitrate transporter MtNRT2.1 is required for optimal nodule establishment in Medicago truncatula under low-nitrate conditions and the repression of nodulation under high-nitrate conditions. The NIN-like protein (NLP) MtNLP1 is required for MtNRT2.1 expression and regulation of nitrate uptake/transport under low- and high-nitrate conditions. Under low nitrate, the gene encoding the C-terminally encoded peptide (CEP) MtCEP1 was more highly expressed, and the exogenous application of MtCEP1 systemically promoted MtNRT2.1 expression in a compact root architecture 2 (MtCRA2)-dependent manner. The enhancement of nodulation by MtCEP1 and nitrate uptake were both impaired in the Mtnrt2.1 mutant under low nitrate. Our study demonstrates that nitrate uptake by MtNRT2.1 differentially affects nodulation at low- and high-nitrate conditions through the actions of MtCEP1 and MtNLP1.

Vacuolar nitrate efflux requires multiple functional redundant nitrate transporter in Arabidopsis thaliana.

Nitrate in plants is preferentially stored in vacuoles; however, how vacuolar nitrate is reallocated and to which biological process(es) it might contribute remain largely elusive. In this study, we functionally characterized three nitrate transporters NPF5.10, NPF5.14, and NPF8.5 that are tonoplast-localized. Ectopic expression in Xenopus laevis oocytes revealed that they mediate low-affinity nitrate transport. Histochemical analysis showed that these transporters were expressed preferentially in pericycle and xylem parenchyma cells. NPF5.10, NPF5.14, and NPF8.5 overexpression significantly decreased vacuolar nitrate contents and nitrate accumulation in Arabidopsis shoots. Further analysis showed that the sextuple mutant (npf5.10 npf5.14 npf8.5 npf5.11 npf5.12 npf5.16) had a higher 15NO3-uptake rate than the wild-type Col-0, but no significant difference was observed for nitrate accumulation between them. The septuple mutant (npf5.11 npf5.12 npf5.16 npf5.10 npf5.14 npf8.5 clca) generated by using CRISPR/Cas9 showed essentially decreased nitrate reallocation compared to wild type when exposed to nitrate starvation, though no further decrease was observed when compared to clca. Notably, NPF5.10, NPF5.14, and NPF8.5 as well as NPF5.11, NPF5.12, and NPF5.16 were consistently induced by mannitol, and more nitrate was detected in the sextuple mutant than in the wild type after mannitol treatment. These observations suggest that vacuolar nitrate efflux is regulated by several functional redundant nitrate transporters, and the reallocation might contribute to osmotic stress response other than mineral nutrition.

The nitrate transporter OsNPF7.9 mediates nitrate allocation and the divergent nitrate use efficiency between indica and japonica rice.

Nitrate allocation in Arabidopsis (Arabidopsis thaliana) represents an important mechanism for mediating plant environmental adaptation. However, whether this mechanism occurs or has any physiological/agronomic importance in the ammoniphilic plant rice (Oriza sativa L.) remains unknown. Here, we address this question through functional characterization of the Nitrate transporter 1/Peptide transporter Family (NPF) transporter gene OsNPF7.9. Ectopic expression of OsNPF7.9 in Xenopus oocytes revealed that the gene encodes a low-affinity nitrate transporter. Histochemical and in-situ hybridization assays showed that OsNPF7.9 expresses preferentially in xylem parenchyma cells of vasculature tissues. Transient expression assays indicated that OsNPF7.9 localizes to the plasma membrane. Nitrate allocation from roots to shoots was essentially decreased in osnpf7.9 mutants. Biomass, grain yield, and nitrogen use efficiency (NUE) decreased in the mutant dependent on nitrate availability. Further analysis demonstrated that nitrate allocation mediated by OsNPF7.9 is essential for balancing rice growth and stress tolerance. Moreover, our research identified an indica-japonica divergent single-nucleotide polymorphism occurring in the coding region of OsNPF7.9, which correlates with enhanced nitrate allocation to shoots of indica rice, revealing that divergent nitrate allocation might represent an important component contributing to the divergent NUE between indica and japonica subspecies and was likely selected as a favorable trait during rice breeding.

Two NPF transporters mediate iron long-distance transport and homeostasis in Arabidopsis.

Iron (Fe) transport and reallocation are essential to Fe homeostasis in plants, but it is unclear how Fe homeostasis is regulated, especially under stress. Here we report that NPF5.9 and its close homolog NPF5.8 redundantly regulate Fe transport and reallocation in Arabidopsis. NPF5.9 is highly upregulated in response to Fe deficiency. NPF5.9 expresses preferentially in vasculature tissues and localizes to the trans-Golgi network, and NPF5.8 showed a similar expression pattern. Long-distance Fe transport and allocation into aerial parts was significantly increased in NPF5.9-overexpressing lines. In the double mutant npf5.8 npf5.9, Fe loading in aerial parts and plant growth were decreased, which were partially rescued by Fe supplementation. Further analysis showed that expression of PYE, the negative regulator for Fe homeostasis, and its downstream target NAS4 were significantly altered in the double mutant. NPF5.9 and NPF5.8 were shown to also mediate nitrate uptake and transport, although nitrate and Fe application did not reciprocally affect each other. Our findings uncovered the novel function of NPF5.9 and NPF5.8 in long-distance Fe transport and homeostasis, and further indicated that they possibly mediate nitrate transport and Fe homeostasis independently in Arabidopsis.

Galactosylation of rhamnogalacturonan-II for cell wall pectin biosynthesis is critical for root apoplastic iron reallocation in Arabidopsis.

Apoplastic iron (Fe) in roots represents an essential Fe storage pool. Reallocation of apoplastic Fe is of great importance to plants experiencing Fe deprivation, but how this reallocation process is regulated remains elusive, likely because of the highly complex cell wall structure and the limited knowledge about cell wall biosynthesis and modulation. Here, we present genetic and biochemical evidence to demonstrate that the Cdi-mediated galactosylation of rhamnogalacturonan-II (RG-II) is required for apoplastic Fe reallocation. Cdi is expressed in roots and up-regulated in response to Fe deficiency. It encodes a putative glycosyltransferase localized to the Golgi apparatus. Biochemical and mass spectrometry assays showed that Cdi catalyzes the transfer of GDP-L-galactose to the terminus of side chain A on RG-II. Disruption of Cdi essentially decreased RG-II dimerization and hence disrupted cell wall formation, as well as the reallocation of apoplastic Fe from roots to shoots. Further transcriptomic, Fourier transform infrared spectroscopy, and Fe desorption kinetic analyses coincidently suggested that Cdi mediates apoplastic Fe reallocation through extensive modulation of cell wall components and consequently the Fe adsorption capacity of the cell wall. Our study provides direct evidence demonstrating a link between cell wall biosynthesis and apoplastic Fe reallocation, thus indicating that the structure of the cell wall is important for efficient usage of the cell wall Fe pool.

[Isolation and characterization of cadmium tolerant gene SpMT2 in the hyperaccumulator Sedum plumbizincicola].

Hyperaccumulators can hyper-accumulate and -tolerate heavy metals, thus are not only an ideal model to explore the mechanisms of ion transport and toxicity tolerance, but also play an irreplaceable role in the development and application of phytoremediation. Sedum plumbizincicola is a recently identified cadmium (Cd)/zinc (Zn) hyperaccumulator in the Crassulaceae family in China. Here we report the construction and screening of its yeast-expressing cDNA library. We identified a metallothionein protein encoding gene SpMT2. SpMT2 is localized in yeast cytoplasm and expression of it in yeast specifically enhanced resistance to Cd. Further analysis showed that SpMT2 did not affect Cd absorption in yeast, but greatly inhibited Cd transport into vacuoles, indicating that SpMT2 may reduce Cd toxicity via chelation in cytoplasm. qRT-PCR analyses indicated that SpMT2 was highly expressed both in roots and shoots, and did not respond to Cd treatment. Taking together the results that SpMT2 was also cytoplasm-localized in plants, we proposed that SpMT2 may chelate/detoxify Cd and retain the complex in cytosol, which renders higher mobility of Cd thus promoting long-distance Cd transport in S. plumbizincicola.

OsProT1 and OsProT3 Function to Mediate Proline- and γ-aminobutyric acid-specific Transport in Yeast and are Differentially Expressed in Rice (Oryza sativa L.).

BACKGROUND: Proline (Pro) and γ-aminobutyric acid (GABA) play important roles in plant development and stress tolerance. However, the molecular components responsible for the transport of these molecules in rice remain largely unknown. RESULTS: Here we identified OsProT1 and OsProT3 as functional transporters for Pro and GABA. Transient expression of eGFP-OsProTs in plant protoplasts revealed that both OsProT1 and OsProT3 are localized to the plasma membrane. Ectopic expression in a yeast mutant demonstrated that both OsProT1 and OsProT3 specifically mediate transport of Pro and GABA with affinity for Pro in the low affinity range. qRT-PCR analyses suggested that OsProT1 was preferentially expressed in leaf sheathes during vegetative growth, while OsProT3 exhibited relatively high expression levels in several tissues, including nodes, panicles and roots. Interestingly, both OsProT1 and OsProT3 were induced by cadmium stress in rice shoots. CONCLUSIONS: Our results suggested that plasma membrane-localized OsProT1 and OsProT3 efficiently transport Pro and GABA when ectopically expressed in yeast and appear to be involved in various physiological processes, including adaption to cadmium stress in rice plants.

The Expected and Unexpected Roles of Nitrate Transporters in Plant Abiotic Stress Resistance and Their Regulation.

Nitrate transporters are primarily responsible for absorption of nitrate from soil and nitrate translocation among different parts of plants. They deliver nitrate to where it is needed. However, recent studies have revealed that nitrate transporters are extensively involved in coping with adverse environmental conditions besides limited nitrate/nitrogen availability. In this review, we describe the functions of the nitrate transporters related to abiotic stresses and their regulation. The expected and unexpected roles of nitrate transporters in plant abiotic stress resistance will also be discussed.

A defensin-like protein drives cadmium efflux and allocation in rice.

Pollution by heavy metals limits the area of land available for cultivation of food crops. A potential solution to this problem might lie in the molecular breeding of food crops for phytoremediation that accumulate toxic metals in straw while producing safe and nutritious grains. Here, we identify a rice quantitative trait locus we name cadmium (Cd) accumulation in leaf 1 (CAL1), which encodes a defensin-like protein. CAL1 is expressed preferentially in root exodermis and xylem parenchyma cells. We provide evidence that CAL1 acts by chelating Cd in the cytosol and facilitating Cd secretion to extracellular spaces, hence lowering cytosolic Cd concentration while driving long-distance Cd transport via xylem vessels. CAL1 does not appear to affect Cd accumulation in rice grains or the accumulation of other essential metals, thus providing an efficient molecular tool to breed dual-function rice varieties that produce safe grains while remediating paddy soils.

Control of proline accumulation under drought via a novel pathway comprising the histone methylase CAU1 and the transcription factor ANAC055.

Proline plays a crucial role in the drought stress response in plants. However, there are still gaps in our knowledge about the molecular mechanisms that regulate proline metabolism under drought stress. Here, we report that the histone methylase encoded by CAU1, which is genetically upstream of P5CS1 (encoding the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase 1), plays a crucial role in proline-mediated drought tolerance. We determined that the transcript level of CAU1 decreased while that of ANAC055 (encoding a transcription factor) increased in wild-type Arabidopsis under drought stress. Further analyses showed that CAU1 bound to the promoter of ANAC055 and suppressed its expression via H4R3sme2-type histone methylation in the promoter region. Thus, under drought stress, a decreased level of CAU1 led to an increased transcript level of ANAC055, which induced the expression of P5CS1 and increased proline level independently of CAS. Drought tolerance and the level of proline were found to be decreased in the cau1 anac055 double-mutant, while proline supplementation restored drought sensitivity in the anac055 mutant. Our results reveal the details of a novel pathway leading to drought tolerance mediated by CAU1.

Tonoplast-localized nitrate uptake transporters involved in vacuolar nitrate efflux and reallocation in Arabidopsis.

A great proportion of nitrate taken up by plants is stored in vacuoles. Vacuolar nitrate accumulation and release is of great importance to nitrate reallocation and efficient utilization. However, how plants mediate nitrate efflux from vacuoles to cytoplasm is largely unknown. The current study identified NPF5.11, NPF5.12 and NPF5.16 as vacuolar nitrate efflux transporters in Arabidopsis. Histochemical analysis showed that NPF5.11, NPF5.12 and NPF5.16 were expressed preferentially in root pericycle cells and xylem parenchyma cells, and further analysis showed that these proteins were tonoplast-localized. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that NPF5.11, NPF5.12 and NPF5.16 were low-affinity, pH-dependent nitrate uptake transporters. In npf5.11 npf5.12 npf5.16 triple mutant lines, more root-fed 15NO3- was translocated to shoots compared to the wild type control. In the NPF5.12 overexpression lines, proportionally less nitrate was maintained in roots. These data together suggested that NPF5.11, NPF5.12 and NPF5.16 might function to uptake nitrate from vacuoles into cytosol, thus serving as important players to modulate nitrate allocation between roots and shoots.

Enhanced metal tolerance correlates with heterotypic variation in SpMTL, a metallothionein-like protein from the hyperaccumulator Sedum plumbizincicola.

Mechanistic insight into metal hyperaccumulation is largely restricted to Brassicaceae plants; therefore, it is of great importance to obtain corresponding knowledge from system outside the Brassicaceae. Here, we constructed and screened a cDNA library of the Cd/Zn hyperaccumulator Sedum plumbizincicola and identified a novel metallothionein-like protein encoding gene SpMTL. SpMTL showed functional similarity to other known MT proteins and also to its orthologues from non-hyperaccumulators. However, three additional cysteine residues were observed in SpMTL and appeared to be hyperaccumulator specific. Removal of these three residues significantly decreased its ability to tolerate Cd and the stoichiometry of Cd against SpMTL (molar ratio of Cd/SpMTL) to a level comparable to those of Cd/SaMTL and Cd/SeMTL in the corresponding non-hyperaccumulating relatives. SpMTL expressed in S. plumbizincicola roots at a much higher level than those of its orthologues in the non-hyperaccumulator roots. Interestingly, a positive correlation was observed between transcript levels of SpMTL in roots and Cd accumulation in leaves. Taking these results together, we propose that elevated transcript levels and heterotypic variation in protein sequences of SpMTL might contribute to the trait of Cd hyperaccumulation and hypertolerance in S. plumbizincicola.

Arabidopsis NRT1.5 Mediates the Suppression of Nitrate Starvation-Induced Leaf Senescence by Modulating Foliar Potassium Level.

Nitrogen deficiency induces leaf senescence. However, whether or how nitrate might affect this process remains to be investigated. Here, we report an interesting finding that nitrate-instead of nitrogen-starvation induced early leaf senescence in nrt1.5 mutant, and present genetic and physiological data demonstrating that nitrate starvation-induced leaf senescence is suppressed by NRT1.5. NRT1.5 suppresses the senescence process dependent on its function from roots, but not the nitrate transport function. Further analyses using nrt1.5 single and nia1 nia2 nrt1.5-4 triple mutant showed a negative correlation between nitrate concentration and senescence rate in leaves. Moreover, when exposed to nitrate starvation, foliar potassium level decreased in nrt1.5, but adding potassium could essentially restore the early leaf senescence phenotype of nrt1.5 plants. Nitrate starvation also downregulated the expression of HAK5, RAP2.11, and ANN1 in nrt1.5 roots, and appeared to alter potassium level in xylem sap from nrt1.5. These data suggest that NRT1.5 likely perceives nitrate starvation-derived signals to prevent leaf senescence by facilitating foliar potassium accumulation.

LeNRT2.3 functions in nitrate acquisition and long-distance transport in tomato.

Nitrogen plays an important role in plant growth and development. Nitrate transporters have been extensively studied in Arabidopsis, but in tomato they have not been functionally characterized. In this study, we report the functions of LeNRT2.3 in nitrate transport in tomato. Our results show that LeNRT2.3 is induced by nitrate, and mainly localizes to the plasma membranes of rhizodermal and pericycle cells in roots. Further analysis in Xenopus oocytes showed that LeNRT2.3 mediates low-affinity nitrate transport. 35S:LeNRT2.3 increased nitrate uptake in root and transport from root to shoot. More interestingly, 35S:LeNRT2.3 showed high biomass and fruit weight. Taken together, these results suggest that LeNRT2.3 plays a double role in nitrate uptake and long-distance transport in tomato.

The Arabidopsis ethylene/jasmonic acid-NRT signaling module coordinates nitrate reallocation and the trade-off between growth and environmental adaptation.

Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.