| The central Shaanxi province is the main district of kiwifruit production in China and in the world,but the soil of this area is calcareous,which can often cause iron(Fe)deficiency chlorosis of kiwifruit leaves due to high bicarbonate content in the soil.At present,bicarbonate-induced Fe deficiency(Bic)has become one of the main bottlenecks restricting the green and sustainable development of the kiwifruit industry in this region.Therefore,The tolerance of different kiwifruit genotypes to Bic were firstly evaluated,and the underlying tolerance mechanism was discussed.Secondly,tissue-cultured seedlings of kiwifruit ‘Qihong’ was used to explore the mechanism of kiwifruit roots in response to Bic.Additionally,the reference genes suitable for gene expression analysis in various parts of different kiwifruit species under iron deficiency conditions were identified.This study provides an important theoretical basis for the analysis of the mechanism of kiwifruit response to induced iron deficiency and the selection of tolerant rootstocks.The main research results were as follows:1.Evaluation of the tolerance of different kiwifruit genotypes to Bic.Taking 11 kiwifruit genotypes from 4 species as tested materials,iron deficiency stress was induced under substrate culture conditions,and 29 growth-and physiology-related indexes were measured.First,the dimensions of the measured indicators were reduced to 9 by ANOVA and correlation analyses;then,the weight of each selected indicator was calculated by principal component analysis,and the comprehensive tolerance score of each genotype was calculated by the weight-based membership function method.In the end,cluster analysis was used to classify its tolerance.The 11 genotypes were divided into 3 categories:(1)tolerance,‘Nongdayuxiang’,Av-1,Acd,Ap,Av-2 and ‘Qinmei’,(2)moderate tolerance:Av-3,Am,Av-4 and ‘Hayward’,(3)sensitive: ‘Qihong’.2.Mechanism of the tolerance difference in two tolerance-contrasting genotypes to Bic.The sensitive kiwifruit cultivar ‘Qihong’ and the tolerant cultivar ‘Nongdayuxiang’were treated by Bic.Compared with ‘Qihong’,Bic resulted in smaller reductions in fresh weight,total Fe and extractable Fe content of ‘Nongdayuxiang’.Compared with‘Nongdayuxiang’,the Fe absorption and transport capacity of the root system of ‘Qihong’was weaker under Bic treatment,resulting in more Fe accumulation in the stem of the plant.In addition,the difference in tolerance to bicarbonate between the two varieties may also be related to the root antioxidant capacity,rhizosphere acidification and differential expression of genes on Fe uptake and transport.3.The mechanism of kiwifruit root in response to Bic.Using kiwifruit ‘Qihong’tissue-cultured seedlings as the test material,the causes of chlorosis in kiwifruit leaves and the characteristics of iron accumulated in roots under the condition of induced iron deficiency were studied using a hydroponic system,and the RNA-seq technology was used to analyze the gene expression in kiwifruit roots under different Fe deficiency conditions.The results showed that bicarbonate was the main cause of leaf chlorosis induced by Fe deficiency in kiwifruit,while the main effect of high pH was to inhibit plant growth.Under bicarbonate stress,active Fe in kiwifruit roots accumulated when Fe-EDTA was used as Fe source,but did not when Fe-EDDHA was used as Fe source.Under bicarbonate stress with Fe-EDTA as Fe source,Fe accumulated in the root apoplast,cell wall hemicellulose and cellulose,and the main forms of Fe accumulation were inorganic form,water-soluble form,phosphoric acid form and oxalic acid form.The amount of differentially expressed genes in roots between Bic and direct Fe deficiency were 2884,including 1819 up-regulated genes and 1062 down-regulated genes.These genes were mainly involved in five KEGG pathways: glutathione metabolism,starch and sucrose metabolism,plant-pathogen interaction,amino sugar and nucleotide sugar metabolism,and plant hormone signal transduction.4.Identification of reference genes for RT-qPCR analysis across kiwifruit species under Fe deficiency conditions.Using 5 kiwifruit genotypes from 4 species as tested materials,the applicability of 10 candidate reference genes in kiwifruit species under direct Fe deficiency and Bic was evaluated.The expression stability of these candidate genes was evaluated by four statistical algorithms(ge Norm,Norm Finder,Best Keeper and Ref Finder).Based on the comprehensive ranking,TCTP ± UBE2 V were best reference genes for roots,UBE2 V ± ACT for stems,RPL19 ± ARF for leaves and UBE2 V ± RPL19 for all the tissues.In addition,6 target genes related to Fe absorption and transport were used to verify the reliability of the recommended reference genes. |
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