Reinforced effect of iron and nitrogen in the process of chlorotic leaves regreening of ‘Dangshansuli’ pear

Abstract:【Objective】China has the largest pear cultivation area in the world. In 2017, the total cultivat- ed area was about 960 000 hm2, and the total output was about 16.53 million tons, accounting for 69.1% and 68.4% of the total cultivated area and total output of the world，respectively.‘Dangshansuli’pear (Pyrus bretschneideri Rehd.) is the largest cultivated variety of pear tree in China, and it was extensive- ly cultivated in the largest region of continuous cultivation, The Old Yellow River Course Region, in Anhui province. In recent years, the phenomenon of iron-deficiency chlorosis in the local areas was carried out.【Methods】The chlorotic and normal sample trees of‘Dangshansuli’pear were used for the ex- periment in the Horticulture Farm of Dangshan County, Anhui province in May 2020, and the following treatments were carried out by using a powered backpack sprayer during the growth period with one non-sprayed tree left as a buffer between the teated trees: (1) CK, normal leaf (N) and chlorotic leaf (C) treated with distilled water; (2) T1, 0.05% FeSO4 solution; (3) T2, 0.10% FeSO4 solution; (4) T3, 0.15% FeSO4 solution; (5) T4, 0.20% FeSO4 solution. Three replicate experiments were adopted for each treatment, and each single experimental unit consisted of three replicate treestrees. All trees were rela- tively consistent in tree shape and vigor, spaced at 4 m×6 m, with seamless crops between the rows, training with the open center system, and the orchard was managed as usual. The retrieved green status was observed on the 4th, 8th and 12th day after treatments, the leaf samples were taken back to the labo- ratory and quickly frozen in liquid nitrogen and stored in the refrigerator at -80 °C for further analysis. The content of total Chlorophyll in the leaf was determined by 80% acetone extraction; the contents of total Fe and Fe2+ were respectively measured by the inductively coupled plasma-atomic emission spec-tometry (ICP-AES) and the phenanthroline colorimetric method; the content of N was determined by micro-kjeldahl method; the chloroplast ultra structure was observed under electron microscope; the total RNA of leaf samples was extracted by the RNAprep Pure Plant Plus Kit (TIANGEN Biochemical Tech- nology Co. Ltd., Beijing, P.R. of China), the cDNA was synthesized using a fluorescence reverse tran- scription kit (TaKaRa Biotechnology Co. Ltd., Dalian, P.R. of China) and the related genes expression of Fe and N metabolism was analyzed by qRT-PCR conducted on an AB StepOne RT-PCR cycler (Ap- plied Biosystems, Foster City, CA, USA); and the related enzymes activity of N metabolism was detect- ed by the ultraviolet spectrophotometry colorimetry.【Results】The more retrieved green spots was formed with 0.05% and 0.10% FeSO4, and the more retrieved green patches formed with 0.15% and 0.20% FeSO4 on the 12th day, and the retrieved green effect of the 0.20% FeSO4 was the most obvious, but the leave color was not as deeply green as the normal leaves. Compared to chlorotic leaves, FeSO4treatment significantly increased the contents of total Chl, total Fe, Fe2+ and N in retrieved green leaves, but the contents of total Chl and N were still significantly lower than those in normal leavesleaves. On the 12th day after treatment with 0.20% FeSO4, the grana lamellae of thylakoid in the leaf were clearer than those of chlorotic leaves, but compared with the normal leaves there were still broken grana lamellae, the chlorotics leaves treated with FeSO4 solution could recover green color to a certain extent, but not completely. The relative expression of FER1, FER2, FER3 and FER4 on the 8th and 12th day after FeSO4 treatment was not only significantly higher than that in the chlorotic leaves, but also significantly higher than that in the normal leaves; the relative expression of FD1 and FD2 was also significantly higher than that in the chlorotic leaves. On the 4th and 8th day, the relative expression of IRT treated with 0.10%, 0.15% and 0.20% FeSO4 was significantly higher than that in the chlorotic leaves. The rela- tive expression of NR, NIR, GLIE and NADH-GOGAT genes with FeSO4 treatment increased significant- ly than that with chlorotic leaves, and the relative enzymes activities were also significantly higher.【Conclusion】Therefore, the chlorotic leaves showed a certain degree of retrieved green color, but still could not completely retrieve green color after application of FeSO4 in different concentrations. The ex- ogenous FeSO4 not only promoted the storage, reduction and transport of iron in chlorotic leaves, but also promoted the reduction of nitric acid and nitrite, to synthesize the glutamic acid and glutamine. The results revealed that iron and nitrogen had a certain synergistic effect in retrieved green process of the chlorotic leaves in‘Dangshansuli’pear.