| ’Hongmian miyou’[Citrus grandis(L.)Osbeck cv.’Hongmian Miyou’]is mutated from ’Guanxi Miyou’,with a light red color,which accumulates lycopene and β-carotene in the spongy layer.It has been approved as a new pummelo cultivar by Fujian Crop Varieties Committee and Sichuan Crop Variety Certification Committee(No.2011008 and No.2016016,respectively).Our team introduced more than ten bud mutants to Sichuan citrus producing areas.After years of field investigation and quality analysis,the results showed ’Hongmian Miyou’ have high commodity and popularization value.Trifoliate orange(Poncirus trifoliata)has been widely used as rootstocks in ’Guanxi miyou’ tree grafting,whereas’Hongmian miyou’ is incompatible with available trifoliate orange rootstocks.Until now,no studies have been reported about the incompatible grafting phenomenon.Grafting is an ancient agricultural practice to propagate commercial fruit species and help plants to overcome their limiting factors in growth.In contrast to the increasing number of studies for the interactions between rootstocks and scions in fruit crops,the mechanism for graft incompatibility is still limited.In this study,we used’Hongmian miyou’ and ’Guanxi miyou’ as scions,trifoliate orange and’Pujiang xiangcheng’ as rootstocks.We measured physiological and biochemical indexes and observed anatomical differences among different graft unions.Comparative transcriptomic analysis was also performed at different stages of graft incompatibility/compatibility combinations to gain further understanding of the reasons for the etiolation of leaves of’Hongmian Miyou’ grafted onto trifoliate orange.The mian results are listed as follows.(1)To determine the graft union formation in citrus,the growth situation of the graft compatibility/incompatibility was recorded.Field investigation showed that there was no significant difference in growth until summer branches matured.Then,the leaves on the summer branches of Hm/Pt turned yellow.Meanwhile,there were obvious decreases for the chlorophyll content and the photosynthesis activity of the leaves.The highest net photosynthesis rate(Pmax)in Hm/Pt,Hm/Cj and Gx/Pt was observed at 98 days after grafting,with the value of 6.57,9.20 and 8.62μmol m-2·s-1,respectively.The photosynthetic rate and chlorophyll content of Hm/Pt were significantly lower than that of Gx/Pt and Hm/Cj.Water content and potential showed that the trend was consistent during whole growth period.The content of sucrose and fructose were significantly increased in graft incompatibility combination.There was no significant difference in glucose content between three graft combinations.The soluble sugar content in incompatibility combination was 1.35 times and 1.17 times than in Hm/Cj and Gx/Pt.According to the histological observation,there were no differences in the anatomical structure between compatible and incompatible combinations.Based on the results obtained from the field performance and anatomical observation,the compatible graft showed higher survival rate than that of the incompatible one but had the same healing process.We belived the yellowing seedlings was caused by abnormal material transport and signal transduction.(2)We also explored the endogenous hormones and mineral elements content in leaf at the developmental stages by HPLC(High Performance Liquid Chromatograph)and AAS(Atomic Absorption Spectrometer)methods.Both major-and micro-elements content of leaves in Hm/Pt kept at the appropriate range,except for excess Mg(0.091 g kg-1 FW)at 404 days after grafting.During the etiolated process,ABA content of Hm/Pt continued to decline,while GA3 content kept at medium level.The level of IAA in Hm/Pt was higher in the early stage,but was significant lower(0.0028～0.0046 mg g-1 FW)in the late stage than that of the control group.Combined with previous studies,we suggested that IAA might play a key role in the process of graft incompatibility.(3)In order to understand the difference between compatible and incompatible grafting combinations,twenty-seven transcriptome libraries from Hm/Pt,Hm/Cj and Gx/Pt grafting combinations were constructed.We found that seven DEGs were significantly differential expressed between graft incompatibility/compatibility combinations.We also found many transcription factores,such as WRKY.KEGG pathway enrichment analysis revealed that DEGs exhibiting differential expression were involved in the chlorophyll metabolism,plant hormone signal transduction and various phytohormones metabolism.(4)We conducted clonging and bioinformatics analysis of promoters of Cg-hmWRKY27 and Cg-gxWRKY27 from Hongmian Miyou and Guanxi Miyou,and analyzed their sequences and expression patterns.Cg-hm WRKY2 7 was expressed in all tissues and showed the highest expression in the spongy layer.Bioinformatics analysis predicted that,the regulatory sequence of Cg-gxWRKY27 and Cg-hmWRKY27 might have a core promoter ranging from 249 to 299,and 250 to 300,with a score of 0.83 and 0.96 and a possible transcriptional initiation site at 290 A and 291 T,respectively.Phylogenetic analysis revealed that CgWRKY27 had closest genetic relationship with that from C.clementina.Promoter core region and transcription start sites were analyzed and no CpG island were predicted in Cg-gxWRKY27 and Cg-hmWRKY27 promoter.There were elements responsive to ABA,ethylene and methyl jasmontae,and anaerobic induction,and defense and stress in both two promoters. |
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