| Arbuscular mycorrhizal(AM) fungus, the ancient phylum Glomeromycota, formed root endosymbiosis with 70%-90% of terrestrial plant species, which increases the uptake of water and minerial nutrients, and remarkably improves the nutritional status of host plants and the resistance against environmental stress. Citrus have rare and short root hairs or even no root-hairs. Therefore roots can not reach the most of rihizosphere soil, which becomes an important limited factor for root absorption of mineral nutrients in soil. So citrus largely depends on AM fungi so as to maintain the normal nutrition level. Citrus is very sensitive to iron deficiency, and the iron deficient problem is frequent in citrus due to the soil environmental reasons. Iron deficiency can increase the contents of phenolic compounds, promoting iron transport in citrus roots. However, information is less unknown on the relationship between the phenolic compounds by AM fungi inducing and iron nutrition under iron deficiency. In this study, the effects of AM fungi on the accumulation, secretion of phenolis and the pal1 gene expression in Poncirus trifoliata L. and Citrus reticulate Blanco inoculated with Glomus versiforme(G.versiforme) were evaluated under iron deficiency stress. The pal1 gene were cloned and bioinformation was analyzed so as to provide basic theory for application of AM fungi on citrus and genetic engineering breeding in citrus. The main results in the present study were as follows:1. The colonization of G.versiforme on P.trifoliata L. and C.reticulate Blanco were study in the pot experiment. The results suggested that the AM colonization rate and hyphal density of P.trifoliata L. and C.reticulate Blanco were 37.5%, 1.6 ± 0.1 m·g-1, and 41.2%, 3.6± 0.1 m·g-1 under iron sufficiency. Those were 29.0%, 2.2 ± 0.3 m·g-1 and 39.5%, 4.1± 0.4 m·g-1 under iron deficiency.2. G.versiforme could significantly increase the growth on P.trifoliata L.and C.reticulate Blanco. Under iron sufficiency, the shoots dry mass, roots dry mass and total biomass of P.trifoliata L. and C.reticulate Blanco seedings inoculated with G.versiforme were 0.58±0.05 g DW/plant, 0.25±0.04 g DW/plant, 0.83±0.08 g DW/plant and 1.18±0.02 g DW/plant, 0.55±0.03 g DW/plant, 1.73±0.08 g DW/plant. Under iron deficiency, those were 0.37±0.01 g DW/plant, 0.21±0.05 g DW/plant, 0.58±0.03 g DW/plant and 1.03±0.01 g DW/plant, 0.47±0.05 g DW/plant, 1.50±0.03 g DW/plant.3. G.versiforme remarkably enhanced the activities of ferric-chelate reductase(FCR) and phenylalnine ammonia-lyase(PAL) in the roots of P.trifoliata L. and C.reticulate Blanco. Under iron sufficiency, the activities of FCR and PAL in the roots of P.trifoliata L. and C.reticulate Blanco seedings inoculated with G.versiforme were 10.47 ?mol·g-1h-1, 120.80 U·g-1FW and 10.32 ?mol·g-1h-1, 167.40 U·g-1FW, which increased by 109.0%, 20.8% and 84.0%, 35.4% compared to control; under iron deficiency, those were 17.63 ?mol·g-1h-1, 138.0 U·g-1FW and 11.32 ?mol·g-1h-1, 296.8 U·g-1FW, which increased by 132.0%, 27.3% and 52.0%, 106.9% respectively.4. G.versiforme could significantly improve the contents of total phenols in root exudates of P.trifoliata L. and C.reticulate Blanco. Under iron sufficiency, the contents of total phenols in root exudates of P.trifoliata L. and C.reticulate Blanco inoculated with G.versiforme increased by 49.3% and 91.8%;under iron deficiency, those increased 44.1% and 44.0%.5. G.versiforme could significantly affect the contents of phenolics in root exudates of P.trifoliata L. and C.reticulate Blanco, however the extent of influence was different between P.trifoliata L. and C.reticulate Blanco. The concents of caffeic acid, chlorogenic acid, cinnamic acid, coumaric acid, gallic acid, protocatechuic and syringic acid in root exudates of P.trifoliata L. inoculated with G.versiforme were increased 3.75 times, 0.33 times, 0.67 times, 0.33 times, 0.5 times, 0.71 times and 0.43 times compared with non-inoculated treatments. Whilst phloretin, phlorizin and salicylic acid were not detected. The concents of coumaric acid, ferulic acid, phlorizin, salicylic acid, syringic acid and vanilloid in root exudates of C.reticulate Blanco inoculated with G.versiforme were increased 1.4 times, 0.67 times, 0.67 times, 2.44 times, 0.71 times and 1.0 times compared with non-inoculated treatments, but caffeic acid, cinnamic acid, phloretin and vanillin were not detected.6. G.versiforme could significantly changed the contents of desorbed Fe from the cell wall by the phenolic compounds in root exudates of P.trifoliata L. and C.reticulate Blanco. The results of desorption kinetic showed that excreted phenolics efficiently desorbed a significant amount of Fe from cell walls, a direct involvement of phenolics in Fe remobilization and phenolics played a key role in improving Fe nutrition of plants.7. Molecular clone and bioinformatics analysis of pal1 gene from citrus roots have been finished. The c DNA sequences of P.trifoliata L. and C.reticulate Blanco were obtained by the method of homology cloning. The bioinformatic analysis indicated that the c DNA sequences of P.trifoliata L. and C.reticulate Blanco were 2166 bp in length, containing a complete open reading frame and 721 amino acids were encoded. The Gene Bank accession number was KF753802 and KP742840. The molecular weight, theoretical p I, formula of the pal1 gene deduced protein of P.trifoliata L. and C.reticulate Blanco were 6.16, 78449.3 Da, C3449H5520N970O1061S28 and 6.09, 78590.4 Da, C3463H5523N969O1062S27. The multiple alignment analysis reveaded that the deduced animo acid sequence of pal1 had typical phenylalanine/histidine ammonia lyase protein tag and included the same conserved deamination sites and conserved catalytic active sites as other plants. Singnal 4.1 analysis indicated there was no signal peptide, so the protein would not be secreted to the outside of the cell. The hydrophobicity analysis showed that the maximum hydrophobicity of the deduced pal1 protein was 2.411(Leu 524), while the minimum was-2.656(Gln 353). The phosphorylation sites analysis told that the deduced pal1 protein of P.trifoliata L. and C.reticulate Blanco included 22 Ser, 9 Thr, 6 Tyr and 20 Ser, 9 Thr, 6 Tyr, which were become the phosphorylation sites of protein kinase.The secondary structure analysis suggested that the deduced pal1 protein of P.trifoliata L. and C.reticulate Blanco mainly included Alpha helix, Random coil, Extended strand, Beta turn, which were 49.51%, 28.57%, 11.79%, 10.12% and 48.13%, 28.99%, 13.04%, 9.85%. The 3D structural modeling of the deduced pal1 protein of P.trifoliata L. and C.reticulate Blanco were performed using SWISS-MODEL, using the Petroselinum crispum PAL protein structure as the template. The phylogenetic tree analysis showed that all pal1 of Rutaceae clustered into a major category.8. G.versiforme could significantly upregulate the pal1 gene expression of the P.trifoliata L. and C.reticulate Blanco. Under iron sufficiency, the pal1 gene expression of the P.trifoliata L. and C.reticulate Blanco inoculated with G.versiforme were 1.63 and 2.02, which were increased by 63.0% and 102.0%. Under iron deficiency, those were 1.88 and 2.57, which were increased 52.3% and 51.9%. |
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