| Citrus is one of the important fruit crops. In China, the cultivation of these crops brought huge economic benefits to the growers. However, the problem that boron deficiency(BD) caused fruit yield reduction is very serious and it makes lots of economic loss for growers. In Gannan, the BD symptoms inclouding corky split vein in leaf and root necrosis usually occurred in the main local cultivars, ’Newhall’ navel orange(Citrus sinensis) grafted in trifoliate orange(Poncirus trifoliata). These negative growth effects decline the vigour of trees rapidly, and eventually affect fruit yield and quality in the coming years. However, little research has been documented on corky split vein development and root rapid response in B deficient citrus. Considering this serious cultural issue, a large interesting gives us to better understand the mechanisms response to BD in citrus plants. In this study, the omics technologies in combination with anatomical and physiological analysis were used to explore the BD responses in both scion leaf and rootstock root. The major results of the research were as follows:1. Anatomical and physiological analysis of corky split vein in BD navel orange:Corky split vein can develop under long term boron deficient conditions in greenhouse. This symptom only occurs in the upper rather than the lower epidermis,which is called polar symptom or polar exhibition. Based on their changes in morphology,4 stages(BD1-BD4) were defined during the corky split vein development. These stages included a phase(BD1) of being similar with control(CK), a phase(BD2) of veins protruding from the blade, a phase(BD3) of slight corking veins and a phase(BD4) of seriously corky split veins. Anatomical investigation of the corky split vein indicated that the symptom was the result of vascular hypertrophy and the higher tolerance to extrusive effect in spongy tissue than that in palisade tissue was the reason of the polar symptom exhibition.Deformation force surveys of the CK and BD veins showed that force values of100% deformation in BD leaves were far less than that in CK leaves, which may be the reason of the brittle property of BD leaves. A lignin test demonstrated that the lignin content was increased at the BD3 stage but no significant difference between the early stages(BD1 and BD2) and the CK samples. The upper epidermis was destroyed by overgrowing vascular cells, suggesting that slight corking veins in the BD3 phase were caused by wounding-induced lignification in wounded cells and seriously corky split veins in the BD4 phase were a combined effect of wounding-induced lignification andinner vascular tissue exposure. These results suggested that the lignin may be considered as an important material for corky split vein development. Ionomic analysis of CK3 and BD3 leaves showed that long term BD not only decreased its B concentration but also reduced the concentration of other 10 elentments, inclouding P, K, Ca, Mg, Fe, Mn, Cu,Zn, Co and Ni.2. Digital gene expression analysis and mi RNAome analysis of corky split vein in BD navel orange:Digital gene expression analysis was performed to analyze the gene expression profilings of corky split veins at four morphologic phases. It revealed that the expressions of genes associated with cytokinin signal transduction, cell division, vascular development, lignin biosynthesis and photosynthesis in corky split veins were all affected,specifically at the BD3 stage. The expressions of WOL involved in the cytokinin signal transduction pathway were up-regulated at BD1 and BD2 phase of corky split vein development. Furthermore, the expressions of vascular development genes, WOX4 and VND7, were up-regulated at the following BD3 and BD4 phases. Up to 40 genes involeved in lignin biosynthesis pathway were up-regulated. Two mi RNAs, csi-mi R156 b and csi-mi R164, were selected by comparative small RNA profiling. Decreased expression of csi-mi R156 b and csi-mi R164, which leads to the up-regulation of their target genes, SPLs(csi-mi R156b-targeted) and CUC2(csi-mi R164-targeted), may promote vascular cell division and orderless stage transition in old leaves.3. Time-course Ca2+ flow analysis of BD trifoliate orange root:The non-invasive micro-test technology(NMT) was used to investigate time-course Ca2+ flow changes of different trifoliate orange root parts(root tip, meristem zone and elongation zone) under BD. Balance buffer with or without B component significantly affected the test results of Ca2+ absorption rate of all three root parts using the control plants. When the plant roots were under BD conditions, Ca2+ absorption rate of three parts were initially increased and then recovered. These results demonstrated that Ca2+ pathway may be involved in the trifoliate orange root early response under BD.4. Proteomic and phosphoproteomic analysis of trifoliate orange roots regarding early BD response:The proteomic and phosphoproteomic analysis were performed between BD 0h and BD 8h roots samples. The results were shown as follows:(1) Ca2+signaling related protein: 3 Ca2+signaling related proteins were altered, 1 Ca2+binding EF-hand domain protein was up-regulated and the other 2 Ca M binding prteions were highlyphosphorylated;(2) Kinase: 11 kinases were selected, 7 of them were altered in protein level and other 4 kinases were altered in phosphorylation status;(3) Cell proliferation related portein: 2 differentially expressed proteins and 2 differentially expressed phosphoproteins were involeved in cell cycle and stem cell control process, which suggesting that their abundance/activity alteration may be related to the occurrence of BD induced root necrosis;(4) Other porteins: some proteins related to protein translation,transport and degradation were also selected for their BD induced significant changes.These BD induced differentially expressed proteins/phosphoproteins provide important cules for studying early BD response in plants. |
PDF Full Text Request