Mechanisms Of Iron Deficiency Tolerance In Different Citrus Rootstocks

Iron?Fe?deficiency caused by calcareous soil is a serious problem in the cultivation of citrus trees in China.The most common corrective measures for Fe deficiency are not effective,and even contaminate the environment.Selecting and developing Fe-deficiency-tolerant rootstocks could be one of the most effective and environmentally friendly strategy to address the Fe chlorosis issue.In this study,we report that Zhique rootstock shows tolerance to Fe deficiency under calcareous soil conditions,but its mechanism was unknown.Here,to understand possible mechanisms of different citrus rootstock tolerant of Fe deficiency,Fe deficiency tolerant rootstock Zhique and Xiangcheng,and an Fe deficiency sensitive rootstock trifoliate orange were used as samples.The main results as follows:1.The physiological mechanism of Fe deficiency tolerant between Zhique and Trifoliate orange rootstock in calcareous soil In the same orchard with calcareous soil,Satsuma mandarin grafted on Zhique displayed normal growth,but those grafted on the most commonly used rootstock,trifoliate orange,showed typical chlorosis.The soil pH and element results showed that soil of the orchard was alkaline and the soil Fe concentration lower the normal threshold for citrus growth;Leaf nutrient analysis revealed that only Fe was much lower in the leaves of Satsuma mandarin/trifoliate orange than in Satsuma mandarin/Zhique.We could conclude that the chlorosis symptom in the leaves of Satsuma mandarin/trifoliate orange was a result of Fe deficiency.Chl a/b concentration of chlorosis leaves in Satsuma mandarin/trifoliate orange significantly lower than green leaves of Satsuma mandarin/Zhique;within the Satsuma mandarin/trifoliate orange samples,the chloroplast inner membrane is obviously degraded,the thylakoid arrangement is irregular,the starch granules are reduced,the number of plastoglobules and mitochondrias were decreased,in the chlorotic leaves in comparison with the green leaves;there was no differences between Zhique and trifoliate orange in roots of total Fe and cell wall Fe concentration,whereas the ferrous Fe in roots of Zhique was higher than trifoliate orange,and the total Fe,ferrous Fe,and cell wall Fe in leaves of Zhique were higher than trifoliate orange.In conclusion,the Fe-deficiency-tolerant rootstock Zhique appears to preserve sufficient ferrous Fe in roots and leaves.2 The relationship between Fe deficiency tolerance and Fe activation in roots of different citrus rootstocks Physiological changes of three citrus rootstocks?Zhique,Xiangcheng and Trifoliate orange?were studied under the hydroponics condition,including three treatments with Fe?iron adequate+Fe;iron adequate+CaCO₃ and NaHCO₃+Fe+Bic;iron adequate+Vanadate+Fe+V?and three treatments without iron Fe?iron deficiency-Fe;iron deficiency+Ca CO₃ and Na HCO₃-Fe+Bic;iron deficiency+Vanadate-Fe+V?.The results showed that,the degree of the Fe chlorosis in Zhique,Xiangcheng and Trifoliate orange were different under iron Fe deficient treatment.Among them,the leaves in Zhique were slightly chlorosis,and Trifoliate orange showed the most seriously chlorosis.The analysis of the Fe concentration in different tissues of three rootstocks were shown:under the+Fe+V treatment,the total Fe in the leaves of Zhique was significantly higher than that in the Xiangcheng and Trifoliate orange;The total Fe in the stems of Zhique was significantly higher than that of Xiangcheng and trifoliate orange in-Fe+V and-Fe+Bic treatments,whereas the total Fe concentration concentration in stems of Zhique and Xiangcheng were higher that trifoliate orange under the other treatments;Total Fe concentration of roots were no differences among three rootstocks under-Fe+Bic treatment,whereas its concentration concentration in roots of Zhique and Xiangcheng were higher that trifoliate orange under the other treatments.The results of ferrous Fe concentration in leaves,stems and roots were shown:the ferrous Fe concentration in the leaves of Zhique and Xiangcheng were significantly higher than that of the Trifoliate orange under three Fe deficiency treatments;The ferrous Fe concentration in stem of Zhique was higher than Xiangcheng and Trifoliate orange,under-Fe and-Fe+V treatments;In roots,the ferrous Fe of Zhique and Xiangcheng were significantly higher than Trifoliate orange under-Fe and-Fe+V treatments,and just Zhique ferrous Fe was higher than Trifoliate orange under-Fe+Bic treatment.The H⁺secretion in roots of Zhique and Xiangcheng were higher than trifoliate orange under Fe deficiency condition.H⁺flow rate in roots of Zhique and Xiangcheng were higher than Trifoliate orange,and its in roots of Zhique was significantly higher than Xiangcheng and Trifoliate orange under+Fe+Bic treatments.These results indicate that the roots of the Zhique and Xiangcheng has stronger acidifying ability and Fe absorption ability under the alkaline condition;H⁺flow rate results shows to some degree that Zhique has a stronger ability to activate rhizosphere Fe under alkaline conditions.3 Molecular response of Fe deficiency in roots of the different citrus rootstocks Ten H⁺-ATPase family members?CsPH1-10?were cloned in the citrus by homologous sequence.The expression level of CsPH2,CsPH4,CsPH7,CsPH8 and CsPH10 in Zhique were higher than that of Trifoliate orange under alkaline soil condition.Further studies showed that the expression levels of CsPH6 in Zhique,Xiangcheng and Trifoliate orange under+Fe and-Fe treatments were higher than that of other gene members,and the expression in Zhique was higher than Xiangcheng and Trifoliate orange.The expression of CsPH6 in Trifoliate orange was significantly higher than the Zhique and Xiangcheng under the+Fe+V and-Fe+V treatments.In transcriptome analysis,29,298,and 500 differentially expressed genes?DEGs?in response to Fe deficiency were identified in ZQ,XC,and TO,respectively.Many transcription factor and ethylene metabolish genes were detected.Quantitative PCR results were used to further verify the expression levels of these genes.Compared with Fe deficiency-related transcriptome data obtained for other species,including Malus xiaojinensis,Poncirus trifoliate,Medicago truncatula,and Arabidopsis thaliana.We hypothesized that the genes associated with ethylene and cell wall metabolism may be related with the differences in Fe deficiency tolerance of different citrus rootstock.