Heterologous expression of strawberry FaFIT promotes iron uptakes by roots in Arabidopsis thaliana

Abstract:【Objective】Iron is involved in the process of physiological and biochemical reactions in plants. Serious iron deficiency can lead to plant chlorosis and even death. Plants have evolved two ways of iron absorption, called strategy Ⅰ and strategyⅡ, to overcome iron deficiency. Among all plants, strategyⅠ exists in such plants other than graminaceous ones. Basic helix-loop-helix (bHLH) transcription factor regulates plant root uptake of iron. FIT, a bHLH gene, is a key gene regulating iron absorption in roots of strategyⅠ plants. FIT gene has been well studied in Arabidopsis, but it has not been reported in strawberry, so that the regulation mode of strawberry FaFIT gene is not clear. The function of strawberry FaFIT gene will be identified in this paper.【Methods】The differentially expressed bHLH gene maker-Fvb3-4-snap-gene-100.50 was obtained from the transcriptome data of Benihoppe root under iron deficiency stress. Further search found that maker-Fvb3-4-snap-gene-100.50 and AtFIT (Arabidopsis) were homologous genes, so the maker-Fvb3-4-snap-gene-100.50 was named FaFIT. The coding region sequence and promoter sequence of FaFIT were searched in GDR (https://www.rosaceae.org/),and PCR primers were designed in NCBI (https://www.ncbi.nlm.nih.gov/). Total RNA was extracted from the roots of Benihoppe and reverse transcribed for cDNA. The coding sequence of FaFIT gene was cloned with cDNA as template. The promoter sequence of FaFIT gene was cloned with DNA as template. The secondary structure of FaFIT protein was predicted by PredictProtein, the conserved amino acid sequence of FaFIT was analyzed by MEME, and the FaFIT promoter elements were predicted by PlantCare. The Benihoppe strawberry plantlets were treated with iron deficiency, iron excess and high pH. The expression patterns of FaFIT gene under abiotic stress were analyzed by real-time fluorescence quantitative PCR (qRT-PCR). The 35S::FaFIT-GFP fusion overexpression vector was constructed to study the gene function of FaFIT in Arabidopsis. The constructed vector was transferred into the strain of Agrobacterium GV3101, and then was infected into Arabidopsis by the dipping flower method. Arabidopsis positive lines were screened with 1/2 MS medium containing 50 mg ·mL-1 kanamycin, and were screened continuously until T2 seeds were obtained. The positive lines of Arabidopsis were identified by PCR. Strategy I of the iron absorption by plant roots is divided into three steps: 1. P-type H+ - ATP (p-type) ase pumps protons (H+ ) to the rhizosphere to acidify the soil and dissolve Fe3+ ions; 2. Ferric-chelate reductase (FCR) reduces Fe3+ to Fe2+ ; 3. Iron-regulated transporter (IRT) transports Fe2+ into root cells. In Arabidopsis, the genes corresponding to these three enzymes are AtAHA2, AtFRO2 and AtIRT1, respectively. Therefore, we used qRT- PCR to determine the gene expression of AtAHA2, AtFRO2 and AtIRT1 in Arabidopsis positive lines, and enzyme-linked immunosorbent assay kit to determine the enzyme activities of H+ -ATPase (p-type), FCR and IRT. Arabidopsis positive lines grew on 1/2 MS medium with pH=5.8 or pH=8.0 to observe the phenotype of Arabidopsis positive lines.【Results】 The total length of FaFIT coding region was 1020 bp and encoded 339 amino-acids. FaFIT protein had a conserved HLH region and belonged to bHLH transcription factor family. The promoter of FaFIT contained bHLH transcription factor DNA binding element, abscisic acid- response elements and drought- stress response elements, which indicated FaFIT might be induced by upstream bHLH transcription factor, ABA and drought-stress. The expression pattern of FaFIT was analyzed by qRT-PCR. The results showed that FaFIT was mainly expressed in roots and induced by iron-deficiency and high pH value but the expression level of FaFIT was significantly down- regulated under excessive iron stress. Benihoppe showed iron chlorotic symptoms under iron deficiency and high pH stress. Excessive iron stress caused harmful impacts on the Benihoppe seedlings. Agrobacterium-mediated transformation of Arabidopsis thaliana showed that the FaFIT-overexpression lines activated the expression of iron absorption related genes AtAHA2, AtFRO2 and AtIRT1, and increased the enzyme activities of H+ -ATPase (p- type), ferric- chelate reductase (FCR) and iron regulated transporter (IRT). FaFIT- overexpression lines of Arabidopsis thaliana accumulated more Fe2 + on 1/2 MS medium with pH=5.8 and pH=8.0. The results showed that FaFIT was specifically expressed in roots and was induced by iron-deficiency stress and high pH value.【Conclusion】FaFIT was inhibited under excessive iron stress. FaFIT can promote the iron absorption in roots by increasing the enzyme activities of FCR, H+ -ATP (p-type)ase and IRT.