The active Fe chelator proline-2'-deoxymugineic acid enhances peanut yield by improving soil Fe availability and plant Fe status.

Iron (Fe) deficiency restricts crop yields in calcareous soil. Thus, a novel Fe chelator, proline-2'-deoxymugineic acid (PDMA), based on the natural phytosiderophore 2'-deoxymugineic acid (DMA), was developed to solve the Fe deficiency problem. However, the effects and mechanisms of PDMA relevant to the Fe nutrition and yield of dicots grown under field conditions require further exploration. In this study, pot and field experiments with calcareous soil were conducted to investigate the effects of PDMA on the Fe nutrition and yield of peanuts. The results demonstrated that PDMA could dissolve insoluble Fe in the rhizosphere and up-regulate the expression of the yellow stripe-like family gene AhYSL1 to improve the Fe nutrition of peanut plants. Moreover, the chlorosis and growth inhibition caused by Fe deficiency were significantly diminished. Notably, under field conditions, the peanut yield and kernel micronutrient contents were promoted by PDMA application. Our results indicate that PDMA promotes the dissolution of insoluble Fe and a rich supply of Fe in the rhizosphere, increasing yields through integrated improvements in soil-plant Fe nutrition at the molecular and ecological levels. In conclusion, the efficacy of PDMA for improving the Fe nutrition and yield of peanut indicates its outstanding potential for agricultural applications.

AhFRDL1-mediated citrate secretion contributes to adaptation to iron deficiency and aluminum stress in peanuts.

Although citrate transporters are involved in iron (Fe) translocation and aluminum (Al) tolerance in plants, to date none of them have been shown to confer both biological functions in plant species that utilize Fe-absorption Strategy I. In this study, we demonstrated that AhFRDL1, a citrate transporter gene from peanut (Arachis hypogaea) that is induced by both Fe-deficiency and Al-stress, participates in both root-to-shoot Fe translocation and Al tolerance. Expression of AhFRDL1 induced by Fe deficiency was located in the root stele, but under Al-stress expression was observed across the entire root-tip cross-section. Overexpression of AhFRDL1 restored efficient Fe translocation in Atfrd3 mutants and Al resistance in AtMATE-knockout mutants. Knocking down AhFRDL1 in the roots resulted in reduced xylem citrate and reduced concentrations of active Fe in young leaves. Furthermore, AhFRDL1-knockdown lines had reduced root citrate exudation and were more sensitive to Al toxicity. Compared to an Al-sensitive variety, enhanced AhFRDL1 expression in an Fe-efficient variety contributed to higher levels of Al tolerance and Fe translocation by promoting citrate secretion. These results indicate that AhFRDL1 plays a significant role in Fe translocation and Al tolerance in Fe-efficient peanut varieties under different soil-stress conditions. Given its dual biological functions, AhFRDL1 may serve as a useful genetic marker for breeding for high Fe efficiency and Al tolerance.

AhDMT1, a Fe(2+) transporter, is involved in improving iron nutrition and N2 fixation in nodules of peanut intercropped with maize in calcareous soils.

Peanut (Arachis hypogaea L.) is an important legume providing edible proteins and N2 fixation. However, iron deficiency severely reduces peanut growth in calcareous soils. The maize/peanut intercropping effectively improves iron nutrition and N2 fixation of peanut under pot and field conditions on calcareous soils. However, little was known of how intercropping regulates iron transporters in peanut. We identified AhDMT1 as a Fe(2+) transporter which was highly expressed in mature nodules with stronger N2 fixation capacity. Promoter expression analysis indicated that AhDMT1 was localized in the vascular tissues of both roots and nodules in peanut. Short-term Fe-deficiency temporarily induced an AhDmt1 expression in mature nodules in contrast to roots. However, analysis of the correlation between the complex regulation pattern of AhDmt1 expression and iron nutrition status indicated that sufficient iron supply for long term was a prerequisite for keeping AhDmt1 at a high expression level in both, peanut roots and mature nodules. The AhDmt1 expression in peanut intercropped with maize under 3 years greenhouse experiments was similar to that of peanut supplied with sufficient iron in laboratory experiments. Thus, the positive interspecific effect of intercropping may supply sufficient iron to enhance the expression of AhDmt1 in peanut roots and mature nodules to improve the iron nutrition and N2 fixation in nodules. This study may also serve as a paradigm in which functionally important genes and their ecological significance in intercropping were characterized using a candidate gene approach.

Lauric acid in crown daisy root exudate potently regulates root-knot nematode chemotaxis and disrupts Mi-flp-18 expression to block infection.

Tomato (Solanum lycopersicum) crops can be severely damaged due to parasitism by the root-knot nematode (RKN) Meloidogyne incognita, but are protected when intercropped with crown daisy (Chrysanthemum coronarium L.). Root exudate may be the determining factor for this protection. An experiment using pots linked by a tube and Petri dish experiments were undertaken to confirm that tomato-crown daisy intercropping root exudate decreased the number of nematodes and alleviated nematode damage, and to determine crown daisy root exudate-regulated nematode chemotaxis. Following a gas chromatography-mass spectrometry assay, it was found that the intercropping protection was derived from the potent bioactivity of a specific root exudate component of crown daisy, namely lauric acid. The Mi-flp-18 gene, encoding an FMRFamide-like peptide neuromodulator, regulated nematode chemotaxis and infection by RNA interference. Moreover, it was shown that lauric acid acts as both a lethal trap and a repellent for M. incognita by specifically regulating Mi-flp-18 expression in a concentration-dependent manner. Low concentrations of lauric acid (0.5-2.0mM) attract M. incognita and consequently cause death, while high concentrations (4.0mM) repel M. incognita. This study elucidates how lauric acid in crown daisy root exudate regulates nematode chemotaxis and disrupts Mi-flp-18 expression to alleviate nematode damage, and presents a general methodology for studying signalling systems affected by plant root exudates in the rhizosphere. This could lead to the development of economical and feasible strategies for controlling plant-parasitic nematodes, and provide an alternative to the use of pesticides in farming systems.

Microbiome convergence enables siderophore-secreting-rhizobacteria to improve iron nutrition and yield of peanut intercropped with maize.

Intercropping has the potential to improve plant nutrition as well as crop yield. However, the exact mechanism promoting improved nutrient acquisition and the role the rhizosphere microbiome may play in this process remains poorly understood. Here, we use a peanut/maize intercropping system to investigate the role of root-associated microbiota in iron nutrition in these crops, combining microbiome profiling, strain and substance isolation and functional validation. We find that intercropping increases iron nutrition in peanut but not in maize plants and that the microbiota composition changes and converges between the two plants tested in intercropping experiments. We identify a Pseudomonas secreted siderophore, pyoverdine, that improves iron nutrition in glasshouse and field experiments. Our results suggest that the presence of siderophore-secreting Pseudomonas in peanut and maize intercropped plays an important role in iron nutrition. These findings could be used to envision future intercropping practices aiming to improve plant nutrition.

Molecular evidence for phytosiderophore-induced improvement of iron nutrition of peanut intercropped with maize in calcareous soil.

Peanut/maize intercropping is a sustainable and effective agroecosystem that evidently enhances the Fe nutrition of peanuts in calcareous soils. So far, the mechanism involved in this process has not been elucidated. In this study, we unravel the effects of phytosiderophores in improving Fe nutrition of intercropped peanuts in peanut/maize intercropping. The maize ys3 mutant, which cannot release phytosiderophores, did not improve Fe nutrition of peanut, whereas the maize ys1 mutant, which can release phytosiderophores, prevented Fe deficiency, indicating an important role of phytosiderophores in improving the Fe nutrition of intercropped peanut. Hydroponic experiments were performed to simplify the intercropping system, which revealed that phytosiderophores released by Fe-deficient wheat promoted Fe acquisition in nearby peanuts and thus improved their Fe nutrition. Moreover, the phytosiderophore deoxymugineic acid (DMA) was detected in the roots of intercropped peanuts. The yellow stripe1-like (YSL) family of genes, which are homologous to maize yellow stripe 1 (ZmYS1), were identified in peanut roots. Further characterization indicated that among five AhYSL genes, AhYSL1, which was localized in the epidermis of peanut roots, transported Fe(III)-DMA. These results imply that in alkaline soil, Fe(III)-DMA dissolved by maize might be absorbed directly by neighbouring peanuts in the peanut/maize intercropping system.

Vanillin in Resistant Tomato Plant Root Exudate Suppresses Meloidogyne incognita Parasitism.

Tomato (Solanum lycopersicum) plants are susceptible to infection by root-knot nematodes, which cause severe economic losses. Planting resistant tomato plants can reduce nematode damage; however, the effects of resistant tomato root exudates in suppressing Meloidogyne incognita remain insufficiently understood. Here, we determined that the resistant tomato plant Lycopersicon esculentum cv. Xianke-8 (XK8) alleviates nematode damage by downregulating the expression of the essential parasitic nematode gene Mi-flp-18 to reduce the infection and reproduction of M. incognita. Using gas chromatography-mass spectrometry, we identified vanillin as a unique compound (compared to susceptible tomato cultivars) in XK8 root exudates that acts as a lethal trap and inhibitor of egg hatching. Moreover, the soil application of 0.4-4.0 mmol/kg vanillin significantly reduced galls and egg masses. The parasite gene Mi-flp-18 was downregulated upon treatment with vanillin, both in vitro and in pot experiments. Collectively, our results reveal an effective nematicidal compound that can use in feasible and economical strategies to control RKNs.

AhNRAMP1 iron transporter is involved in iron acquisition in peanut.

Peanut/maize intercropping is a sustainable and effective agroecosystem to alleviate iron-deficiency chlorosis. Using suppression subtractive hybridization from the roots of intercropped and monocropped peanut which show different iron nutrition levels, a peanut gene, AhNRAMP1, which belongs to divalent metal transporters of the natural resistance-associated macrophage protein (NRAMP) gene family was isolated. Yeast complementation assays suggested that AhNRAMP1 encodes a functional iron transporter. Moreover, the mRNA level of AhNRAMP1 was obviously induced by iron deficiency in both roots and leaves. Transient expression, laser microdissection, and in situ hybridization analyses revealed that AhNRAMP1 was mainly localized on the plasma membrane of the epidermis of peanut roots. Induced expression of AhNRAMP1 in tobacco conferred enhanced tolerance to iron deprivation. These results suggest that the AhNRAMP1 is possibly involved in iron acquisition in peanut plants.

Nematicidal Effect of Methyl Palmitate and Methyl Stearate against Meloidogyne incognita in Bananas.

Banana plants (Musa spp.) are susceptible to infection by many plant-parasitic nematodes, including Meloidogyne incognita. In this study, a mixed fermentation broth of chicken manure (CM) and cassava ethanol wastewater (CEW) was used to inhibit M. incognita by reducing egg hatching and by having a lethal effect on second-stage juvenile nematodes (J2s). It also alleviated nematode damage and promoted banana plant growth. Using gas chromatography-mass spectrometry (GC-MS), we identified methyl palmitate and methyl stearate as bioactive compounds. These bioactive compounds repelled J2s and inhibited egg hatching; reduced root galls, egg masses, and nematodes in soil; and downregulated the essential parasitic nematode genes Mi-flp-18 and 16D10. A Caenorhabditis elegans offspring assay showed that low concentrations of the fermentation broth, methyl palmitate, and methyl stearate were safe for its life cycle. This study explored the effective and environmentally safe strategies for controlling root-knot nematodes.

From Leguminosae/Gramineae Intercropping Systems to See Benefits of Intercropping on Iron Nutrition.

To achieve sustainable development with a growing population while sustaining natural resources, a sustainable intensification of agriculture is necessary. Intercropping is useful for low-input/resource-limited agricultural systems. Iron (Fe) deficiency is a worldwide agricultural problem owing to the low solubility and bioavailability of Fe in alkaline and calcareous soils. Here, we summarize the effects of intercropping systems on Fe nutrition. Several cases showed that intercropping with graminaceous plants could be used to correct Fe nutrition of Leguminosae such as peanut and soybean or fruits such as Psidium guajava L., Citrus, grape and pear in calcareous soils. Intercropping systems have strong positive effects on the physicochemical and biochemical characteristics of soil and the microbial community due to interspecific differences and interactions in the rhizosphere. Rhizosphere interactions can increase the bioavailability of Fe with the help of phytosiderophores. Enriched microorganisms may also facilitate the Fe nutrition of crops. A peanut/maize intercropping system could help us understand the dynamics in rhizosphere and molecular mechanism. However, the role of microbiome in regulating Fe acquisition of root and the mechanisms underlying these phenomena in other intercropping system except peanut/maize need further work, which will help better utilize intercropping to increase the efficiency of Fe foraging.

AhNRAMP1 Enhances Manganese and Zinc Uptake in Plants.

Manganese (Mn) and zinc (Zn) play essential roles in plants. Members of the natural resistance-associated macrophage protein (NRAMP) family transport divalent metal ions. In this research, the function of peanut (Arachis hypogaea L.) AhNRAMP1 in transporting Mn and Zn, as well as its potential for iron(Fe) and Zn biofortification was examined. AhNRAMP1 transcription was strongly induced by Mn- or Zn-deficiency in roots and stems of peanut. Yeast complementation assays suggested that AhNRAMP1 encoded a functional Mn and Zn transporter. Exogenous expression of AhNRAMP1 in tobacco and rice enhanced Mn or Zn concentrations, improving tolerance to Mn or Zn deficiency. With higher Mn concentration, transgenic plants exhibited inhibited growth under Mn excess condition; similar results were obtained under excessive Zn treatment. AhNRAMP1 expression increased biomass in transgenic tobacco and rice, as well as yield in transgenic rice grown on calcareous soil. Compared with non-transformed (NT) plants, Fe and Zn concentrations were elevated whereas concentrations of Mn, copper (Cu), and cadmium (Cd) were not enhanced. These results revealed that AhNRAMP1 contributes to Mn and Zn transport in plants and may be a candidate gene for Fe and Zn biofortification.

Comparative transcriptomic analysis of the roots of intercropped peanut and maize reveals novel insights into peanut iron nutrition.

Intercropping is a vital technology in resource-limited agricultural systems with low inputs. Peanut/maize intercropping enhances iron (Fe) nutrition in calcareous soil. In this study, the transcriptome of peanut and maize roots was analyzed by suppression subtractive hybridization (SSH) and microarray analysis separately. We constructed four SSH libraries using the cDNA of peanut roots based on two cropping patterns: monocropping and intercropping, and two growth stages: vegetative stage and reproductive stage. Lib M1, I1, M2 and I2 comprised 53, 51, 37 and 54 genes, respectively. Six and four transporters were found in the two intercropping-specific SSH libraries, which may facilitate Fe acquisition and protoplasmic homeostasis of metal ions and anions. Specifically, AhNARMP1 and MTP may play a role in boosting Fe nutrition during the vegetative stage. The expression of MYC2 was also upregulated by intercropping, while an ethylene-responsive transcription factor was downregulated during two growth periods. Microarrays indicated that homocysteine S-methyltransferase and serine acetyltransferase 1 upregulated in intercropped maize roots, which directly associated with methionine biosynthesis. It may account for the enhanced phytosiderophore released capacity in intercropping, which benefited the Fe nutrition of intercropped peanut in reproductive stage. Two aminocyclopropane-1-carboxylic acid synthase oxidase genes, which are related to ethylene biosynthesis, were downregulated in maize root by intercropping. Taken together with our previous proteomic work, the results indicated that intercropping enhances jasmonate signaling and weakens ethylene signaling in peanut and maize roots, which may improve ecological adaptation of the peanut plant to intercropping systems.

The Yellow Stripe-Like (YSL) Gene Functions in Internal Copper Transport in Peanut.

Copper (Cu) is involved in fundamental biological processes for plant growth and development. However, Cu excess is harmful to plants. Thus, Cu in plant tissues must be tightly regulated. In this study, we found that the peanut Yellow Stripe-Like family gene AhYSL3.1 is involved in Cu transport. Among five AhYSL genes, AhYSL3.1 and AhYSL3.2 were upregulated by Cu deficiency in peanut roots and expressed mainly in young leaves. A yeast complementation assay suggested that the plasma membrane-localized AhYSL3.1 was a Cu-nicotianamine complex transporter. High expression of AhYSL3.1 in tobacco and rice plants with excess Cu resulted in a low concentration of Cu in young leaves. These transgenic plants were resistant to excess Cu. The above results suggest that AhYSL3.1 is responsible for the internal transport of Cu in peanut.

Effects of Fe-deficient conditions on Fe uptake and utilization in P-efficient soybean.

Phosphorus (P)-efficient soybean (Glycine max) plants absorb and utilize P with high efficiency. To investigate the effects of iron (Fe)-deficient conditions on the absorption and utilization of Fe in P-efficient soybean plants, two soybean cultivars with different P efficiency, the 03-3 (P-efficient variety) and Bd-2 (P-inefficient variety), were used in this study. The two soybean cultivars were grown in nutrient solution containing Fe concentrations of 0 (Fe0), 20 (Fe20), 40 (Fe40), or 80 (Fe80) µM for 7 days. The Fe reductase activity of roots was higher in 03-3 plants grown under the Fe0, Fe20, and Fe40 treatments than in Bd-2 plants and the total Fe uptake was greater in 03-3 plants under the Fe40 treatment. GmFRD3a was much more highly expressed in the stem of 03-3 than in that of Bd-2, and significantly more iron was transported to 03-3 plant shoots during Fe0 treatment. Chlorosis in young leaves caused by Fe deficiency under the Fe0 and Fe20 treatments was alleviated by increased Fe concentration in shoots. Increased levels of active Fe in young 03-3 leaves under Fe-deprivation conditions (Fe0) and maintenance of stable Fe concentrations in 03-3 shoots subjected to Fe20, Fe40, and Fe80 treatments suggested that the P-efficient 03-3 cultivar is also Fe-efficient. It is suggested that 03-3 soybean cultivar should be a good resource for application to farm field.

Mi-flp-18 and Mi-mpk-1 Genes are Potential Targets for Meloidogyne incognita Control.

Meloidogyne incognita is a major plant parasite that causes root-knot disease in numerous agricultural crops. This nematode has severely affected greenhouse crops in China. Chemical insecticides are generally used to control this pest, but they have adverse environmental and human toxicity effects; hence, safe and effective strategies for controlling the root-knot nematode (RKN) are necessary. FMRFamide-like peptides (FLPs) have diverse physiological and biological effects on the locomotory, feeding, and reproductive functions of nematodes, and mitogen-activated protein (MAP) kinase plays an important role in the regulation of transcription factors and protein kinases. These candidates are the common targets of RKN control. They are encoded by Mi-flp-18 and Mi-mpk-1 genes, respectively, in M. incognita . In this study, we used the RNA interference (RNAi) method to silence the transcription of these genes and determined the effects on the pathogenicity of RKN in potted plants. Real-time quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that Mi-mpk-1 gene expression could be reduced by 33% by RNAi. The RNAi-treated infective nematodes were inoculated with dsRNAs of Mi-flp-18 and Mi-mpk-1 in pot experiments. The root-knot numbers were reduced by 51% after Mi-flp-18 RNAi treatment. Further, the relative abundance of Mi-flp-18 was downregulated by 79% in the endoparasitic M. incognita . Mi-flp-18 RNAi treatment decreased egg masses by 92% and egg numbers by 58%. Mi-mpk-1 RNAi treatment reduced the root-knot numbers by 32% and, remarkably, lowered the relative abundance of Mi-mpk-1 in the endoparasitic M. incognita . Egg masses and numbers were reduced by 42 and 22%, respectively, after RKN was inoculated for 35 days with Mi-mpk-1 RNAi. Therefore, Mi-flp-18 and Mi-mpk-1 genes are susceptible to RNAi and can be used as potential targets for RKN control by regulating nematode infection, parasitism, and reproduction.

Dynamics in the rhizosphere and iron-uptake gene expression in peanut induced by intercropping with maize: role in improving iron nutrition in peanut.

The intercropping of maize with peanuts is an effective cropping practice. Indeed, peanut/maize intercropping reportedly improves the iron nutrition of peanuts in calcareous soils. The limited evidence available suggests that the improved Fe nutrition in intercropping is largely attributable to a rhizosphere effect of maize. In this study, the effects of peanut/maize intercropping on the Fe nutritional status of peanut associated with the dynamics of the rhizosphere processes and Fe uptake gene expression induced by the interaction of the two species at various growth days were investigated. The results suggest that an interspecific rhizosphere effect improves Fe nutrition in peanut, as shown by changes in the rhizosphere available Fe concentration, pH, and Olsen-P concentration, based on time-course changes in peanut-maize interaction. The increase in available Fe in the rhizosphere of peanut ranged from 0.2 to 2.64 mg kg(-1). The transition from the vegetative to reproductive stage was a key turning point in the time-course of changes in the rhizosphere processes in intercropping. There was more consistently positive effect of intercropping on peanut Fe nutrition after 53 days. Moreover, the expression of AhFRO1 and AhYSL1 was expressed at significantly higher level in intercropped peanuts compared to monocropped peanut at the vegetative stage, indicating a role for these genes in Fe improvement in intercropped peanuts. We conclude that the enhanced time-course changes in the rhizosphere processes and iron uptake gene expression with a consistent positive interspecific effect appear to be one of the mechanisms underlying the improved Fe nutrition in intercropped peanut plants.

Expression of peanut Iron Regulated Transporter 1 in tobacco and rice plants confers improved iron nutrition.

Iron (Fe) limitation is a widespread agricultural problem in calcareous soils and severely limits crop production. Iron Regulated Transporter 1 (IRT1) is a key component for Fe uptake from the soil in dicot plants. In this study, the peanut (Arachis hypogaea L.) AhIRT1 was introduced into tobacco and rice plants using an Fe-deficiency-inducible artificial promoter. Induced expression of AhIRT1 in tobacco plants resulted in accumulation of Fe in young leaves under Fe deficient conditions. Even under Fe-excess conditions, the Fe concentration was also markedly enhanced, suggesting that the Fe status did not affect the uptake and translocation of Fe by AhIRT1 in the transgenic plants. Most importantly, the transgenic tobacco plants showed improved tolerance to Fe limitation in culture in two types of calcareous soils. Additionally, the induced expression of AhIRT1 in rice plants also resulted in high tolerance to low Fe availability in calcareous soils.

Comparative proteomic analysis for assessment of the ecological significance of maize and peanut intercropping.

Intercropping is an important and sustainable cropping practice in agroecosystems. Peanut/maize intercropping is known to improve the iron (Fe) content of peanuts in calcareous soils. In this study, a proteomic approach was used to uncover the ecological significance of peanut/maize intercropping at the molecular level. We demonstrate that photosynthesis-related proteins accumulated in intercropped peanut leaves, suggesting that the intercropped peanuts had a stronger photosynthetic efficiency. Moreover, stress-response proteins displayed elevated expression levels in both peanut and maize in a monocropping system. This indicated that intercropping contributes to resistance to stress conditions. Allene oxide synthase and 12-oxo-phytodienoic acid reductase, two key enzymes in jasmonate (JA) biosynthesis, increased in abundance in the maize roots of the intercropping system, consistent with the upregulation of JA-induced proteins shown by microarray analysis. These results imply that JA may act as a signaling molecule, playing an important role in intercropping through rhizosphere interaction. This study suggests that peanut/maize intercropping results in high Fe availability in the rhizosphere, leading to variation in the proteins related to carbon and nitrogen metabolism. The advantages of intercropping systems may improve the ecological adaptation of plants to environmental stress.

[Effects of peanut mixed cropping with different gramineous plants on apoplast iron accumulation and reducing capacity of peanut].

The effects of peanut mixed cropping with five different gramineous plants on apoplast iron accumulation and reducing capacity of peanut were investigated by soil culture experiment. The results showed that mixed cropping of maize, barley, oats, wheat, and sorghum with peanut could improve iron nutrition of peanut respectively. The phytosiderophores excretion rate of barley, oats and wheat were much higher than that of maize, and the phytosiderophores excretion rate of sorghum was lower than that of maize. In comparison with peanut in monocropping the iron content in different organs of peanut mixed with maize, barley, oats, wheat and sorghum were increased. The Fe content in root apoplast of peanut mixed with five different gramineous was gradually increased and higher than that of peanut in monocropping at different growth days. At the same time, the mixed cropping systems remarkably improved the soil Fe availability in the rhizosphere and root Fe reducing capacity of peanut. The higher root Fe (III) reducing capacity and much more available Fe in the rhizospe (III) of peanut in mixed cropping played an important role in improving iron nutrition of peanut.