Physiological And Molecular Mechanism Underlying Phosphorus-mediated Manganese Toxicity Alleviation In Peach

Peach（Prunus persica）is a commercially important fruit crop,mostly grown in temperate regions all over the world,with a global production of approximately 25 million tons annually,half of which is produced in China.Particularly,Shuimitao peach,also known as honey or juicy peach,is mostly cultivated in the southern parts of China,where the soil is predominantly acidic.In addition,excessive rainfall in this area and the overuse of fertilization and manganese-containing chemicals（e.g.,maneb and mancozeb）,increases manganese（Mn）availability in acidic soil,which promotes Mn toxicity in plants,and emerged as a significant concern for peach-producing industries.Therefore,addressing such issue is one of the major concerns in peach production areas for the sustainable industry.Mn is a key micronutrient for plant development,and crucial for cellular metabolic processes in plants.Particularly,Mn is an essential constituent of structural proteins and plays a key role in photosynthesis.Furthermore,Mn also plays an important role in secondary metabolism,involved in the activation of enzymes required for monolignols,which are responsible for the biosynthesis of lignin polymers.Despite of its importance,the increased accumulation of Mn in acidic soil has been reported as a considerable obstacle to plant development.As soil p H decreases,the availability of Mn²⁺increases in the soil solution.Therefore,Mn was mainly absorbed by roots as Mn²⁺,and causes Mn toxicity in plants.The acquisition of Mn from soil and long-distance transportation in plants are facilitated by various transporter families.The immediate reactions of plants to Mn toxicity stress are a decrease in photosynthetic rate.Thus,oxidation of Mn in the chloroplast by photo-oxidation of chlorophyll（Chl）generates reactive oxygen species（ROS）,affects Chl,and therefore destroys the chloroplast ultrastructure.Furthermore,Mn²⁺are rapidly absorbed and acquired by roots,which primarily affect plant roots and result in stunted root growth.However,phosphorus（P）is another key nutrient for plant growth and development while participating in several biological processes,including photosynthesis and carbohydrate metabolism.Moreover,P is a part of many key molecules and is essential for cell wall integrity.Although the interaction between P and Mn is observed in a few plant species,the contribution of P in promoting Mn stress tolerance in horticultural crops,such as fruit crops,remains to be elucidated.Therefore,investigations on the physiological and molecular mechanism of P-mediated Mn stress alleviation in peach plants has a great influence on scientific and practical implications for the mitigation of Mn toxicity in peach plants.In the current study,initially we investigated the morphophysiological effects of Mn stress and P-mediated alleviation of Mn toxicity in peach plants.Further,the variations in gene expression of peach plants in response to P-mediated mitigation of Mn stress were studied by using transcriptomic approach.Subsequently,we identified and explored the functional characterization of PpNRAMP5 in response to Mn stress in peach plants.The main findings of the research are as follows:1.The accumulated Mn led to a considerable reduction in plant biomass,water status,Chl content,photosynthetic rate,and disrupted the chloroplast ultrastructure by increasing oxidative stress(H₂O₂ and O₂^·-).However,P supplementation dramatically improved plant biomass,leaf relative water and Chl contents,upregulating the ascorbate-glutathione pool and increasing the activities of antioxidant enzymes（superoxide dismutase;peroxidase dismutase;ascorbate peroxidase;monodehydroascorbate reductase;dehydroascorbate reductase）,thus reducing oxidative damage as evidenced by lowering H₂O₂ and O₂^·-staining intensity.Moreover,P application markedly restored stomatal aperture and improved chloroplast ultrastructure,as indicated by the improved performance of photosynthetic machinery.Altogether,our findings suggest that P has a great potential to induce tolerance against Mn toxicity by limiting Mn accumulation in tissues,upregulating antioxidant defense mechanisms,alleviating oxidative damage,improving chloroplast ultrastructure and photosynthetic performance in peach plants.2.Mn toxicity negatively affect the root architecture,morphology,disrupts the root ultrastructure,also causes numerous cellular structural damages due to disruption in redox state and caused oxidative injury and thus reduced the lignin content.Lignin is essential for the structural integrity,elasticity,and strength of the cell wall in plants,hence provide plant tolerance against heavy metals stress.However,P application improved the roots structure and decreased the ROS accumulation,as evidenced by reducing the green fluorescence intensity of H₂O₂staining and thereby reduced the lipid peroxidation by activating enzymatic antioxidants.A total of 17981 differently expressed genes（DEGs）were identified after P application under Mn toxicity.Additionally,DEGs related to membrane-located Mn transporter genes was markedly downregulated by P application under Mn toxicity.The DEGs mainly enriched in phenylpropanoid pathway,which produces lignin as an end-product in vascular plants.Therefore,P application upregulated the expression of caffeoyl-Co A O-methyltransferase,caffeic acid 3-O-methyltransferase,and guaiacol-peroxidase and as well as their enzyme activities involved in lignin biosynthesis pathway,thus increased the lignin content.Hence,provide strength to the roots by improving cell wall ultrastructure and root growth.Altogether,P regulates the lignin biosynthesis pathway under Mn toxicity,our results further provide in-depth insights into the mechanism of P-mediated alleviation of Mn stress in peach plants.3.The expression profiling analysis indicated that PpNRAMPs were significantly induced by excess Mn,iron,zinc,and cadmium treatments,suggesting their potential role in heavy metal uptake and transportation.Notably,the expression of PpNRAMP5 was tremendously increased under Mn toxicity stress.Heterologous expression of PpNRAMP5 in yeast cell also confirmed Mn transport.Suppression of PpNRAMP5 through virus-induced gene silencing enhanced Mn toxicity tolerance,which was compromised when PpNRAMP5 was overexpressed in peach.The silencing of PpNRAMP5 mitigated Mn toxicity by dramatically reducing Mn contents in roots,and effectively reduced the Chl degradation and improved the photosynthetic apparatus under Mn toxicity stress.Therefore,PpNRAMP5-silenced plants were less damaged by oxidative stress,as signified by lowered H₂O₂ contents and O₂^·-staining intensity,also altered the ROS homeostasis by modulating enzymatic antioxidants.Consistently,these physiological changes showed an opposite trend in PpNRAMP5-overexpressed plants.Altogether,our findings suggest that downregulation of PpNRAMP5markedly reduces the uptake and transportation of Mn,thus activating enzymatic antioxidants to strengthen ROS scavenging capacity and thereby mitigating Mn toxicity in peach plants.These findings highlight the fundamental insights into physiological,biochemical,and transcriptomic approaches to reveal the role of P in Mn-stressed peach plants.Additionally,the present work also provides an understanding of the role of NRAMP gene family in P.persica under Mn toxicity stress.Our study also provides novel insights on the possibility of genetic modifications to improve Mn phytoremediation efficiency.