Regulation Mechanism Of Growth Promotion And Flavonoid Accumulation In Tomato Fruit By Iron-based Nanomaterials

With the rapid development of nanotechnology,nanomaterials（NMs）have been used in agriculture because of their unique properties.NMs can promote seed germination and growth,and increase yield and fruit quality.Flavonoid content is one of the important indicators of fruit quality,which helps to improve fruit nutritional quality.Flavonoids are also beneficial to human health for effectively preventing several chronic diseases and cancers.Therefore,people are more and more interested in the development of flavonoid-rich crops,but the regulation mechanisms of the promotion of flavonoids by NMs remain unclear.As a model berry vegetable,tomato is the second most traded agricultural product in the global market.Increasing the flavonoid content in tomato can help increase the dietary flavonoid intake of consumers,thus promotes human health and prevents diseases.The biosynthesis of flavonoids in plants requires the participation of iron,which is a key element related to the synthesis of flavonoids.Phosphorus acts as a limiting mass element required by plants in soil.In this study,rod-like NMs were successfully prepared by adding dispersant PVP-K30 based on the conventional hydrothermal synthesis method and the chemical formula was determined as Triiron Tetrairon Phosphate（Fe₇（PO₄）₆）.The Fe₇（PO₄）₆ NMs were further coated with FITC and used in the subsequent soil exposure experiments.We conducted seedling,flowering and maturity experiments of tomato plants to explore the regulatory mechanisms of NMs on the growth of seedling,flower development and flavonoid synthesis of fruits at maturity.The main results are as follows:（1）In the preliminary experiments,compared with 5 mg·kg^-1 application and control,50mg·kg^-1 Fe₇（PO₄）₆ NMs had the most relative content of 3 types of flavonoids:naringenin,quercetin and rutin,so 50 mg·kg^-1 was the most suitable concentration and used in subsequent experiments.Tomato seedlings exposed to soil(50 mg kg^-1)showed that Fe-bearing particles were accumulated in roots(4.8×10⁶ particles g^-1)while it was not detected in stems and leaves.No fluorescence from particles was observed in roots which indicated that NMs were accumulated in the root and not transported upward.Fe₇（PO₄）₆ NMs in roots affected the regulation of root hormone signaling and nutrient uptake.Fe₇（PO₄）₆ NMs induced IAA（Indole-3-acetic acid）accumulation and activated LHA1（424.9%compared to control）,the key gene of plasma membrane（PM）H⁺ATPase in roots,which caused PM H⁺ATPase to pump a large amount of H⁺out of roots(5.9 pmol cm^-2 s^-1)and acidify rhizosphere.Fe₇（PO₄）₆ NMs also promoted root growth and absorption of Mn,Ca,K,Mg and Fe,and then enhanced photosynthesis and increased biomass.In addition,Fe₇（PO₄）₆ NMs also significantly enriched and increased the abundance of actinomycetes in rhizosphere.The actinomycetes could promote the plant growth through nutrient uptake.Moreover,compared to control,Fe₇（PO₄）₆NMs significantly up-regulated the expression levels of sucrose transport genes SUT1 and SUT2 in leaves over 44.1%and 34.2%,respectively,suggesting that Fe₇（PO₄）₆ NMs enhanced sucrose transport capacity in tomato seedlings.（2）On the basis of promoting seedling growth,Fe₇（PO₄）₆ NMs still significantly improved tomato photosynthesis at flowering stage.Fe₇（PO₄）₆ NMs enhanced photosynthesis and significantly up-regulated the expression of sucrose transport gene SUT1（2.0 folds compared to control）to promote sucrose transport from leaves into flowers,thus provided more energy for flower development.No Fe-bearing particles were detected in tomato roots treated by Fe₇（PO₄）₆ NMs,and neither was fluorescence from the NMs.These results indicated that Fe₇（PO₄）₆ NMs didn’t remain in roots with plant growth.But Fe₇（PO₄）₆ NMs promoted Ca absorption and accumulation in flowers and then induced ovule development,improved pollen activity and pollen tube germination.（3）The maturity experiments showed that Fe₇（PO₄）₆ NMs significantly enriched more plant growth promotion rhizosphere microorganisms（PGPRs）,which could enhance tomato growth and fruit development,and significantly increased sucrose accumulation in fruit（21.2%compared to control）that provided more precursors and signals for flavonoid synthesis.Under NMs treatment,the contents of naringenin,quercetin and rutin in mature fruits were significantly increased by 6.8,0.6 and 1.2 times compared with the control,reaching 9.6μg 100g FW^-1,30.5 and 3.3 mg 100 g FW^-1 and the results showed that NMs significantly promoted the synthesis and accumulation of flavonoids.Metabolomics and transcriptomics analysis of sucrose metabolism-shikimic acid metabolism-phenylpropanoid metabolic pathway-flavonoid synthesis in fruits showed that the abundance of sucrose products UDP-glucose,trehalose and glucose-6-phosphate（G6P）increased significantly in the sucrose metabolic pathway.Sucrose and trehalose are signals that regulate the synthesis of flavonoids and their increase up-regulates flavonoids synthesis.Transcriptional genes Sl SS,Sl TPS6 and Sl TPP,which regulate the conversion of sucrose to trehalose,and SLTKL-1,which regulates the production of erythrose4-phosphate from G6P,were significantly up-regulated;in shikimic acid metabolic pathway,only Sl DHQ-SOR（a transcription gene encoding bifunctional enzyme catalyzing dehydroquinate to shikimic acid）was detected and significantly down-regulated.Since ABA can inhibit shikimic acid pathway,the increased endogenous ABA content and the activation of ABA response pathway in fruits indicated that shikimic acid metabolic pathway was inhibited by increased ABA;the transcription levels of Sl EPSPS-1（encoding the enzyme catalyzes the formation of EPSP from shikimate-3-phosphate and phosphoenolpyruvate）and Sl CM2（encoding the enzyme rearranges the enolpyruvyl side chain of chorismate to form prephenate）in phenylpropanoid metabolic pathway were significantly down-regulated;in the flavonoid synthesis pathway,the transcription level of the rate-limiting enzyme CHI（chalcone isomerase）and the expression of the transcription factor Sl MYB12 were significantly increased,and the contents of naringin,quercetin and rutin were also significantly accumulated.In summary,this study revealed that Fe₇（PO₄）₆ NMs activated the flavonoids synthesis pathway through up-regulating transcriptional gene expression of sucrose metabolism and flavonoid biosynthesis.