Different Response And Structure Change And Metabolic Mechanism Of Different Citrus Rootstock Under Boron Deficiency

It is well known that boron(B)is a crucial micronutrient required for vascular plants.Citrus,in China,is ranked as a chiefly significant profitable fruit crops.Ganzhou city in Jiangxi province in southern China is a predominant region of navel orange production,and plays an important role in citrus production in China.However,soil in this area contains very low level(<0.25 mg kg-1)of available B.In recent years,B deficiency is frequently observed in citrus orchards,and is responsible for considerable loss of productivity,quality and productivity.Trifoliate orange [Poncirus trifoliata(L.)Raf.,main cultivated variety] and citrange [Citrus sinensis(L.)Osb.× Poncirus trifoliata(L.)Raf.,newly introduced variety] are considered a kind of important rootstock resource.There are different B-deficiency symptoms between citrus grafted on different rootstocks observed in citrus orchards.Therefore,it is significant to carry out research works on the mechanism of B efficiency in trifoliate orange and citrange rootstock,and on the response mechanism of B deficiency on the cell wall structure and metabolism in citrus rootstock.Moreover,this study could provide the scientific basis for B nutrition study of perennial citrus and the fertilizer measure on different rootstocks.In the present study,citrange and trifoliate orange rootstock seedlings were selected as materials,and then exposed to different amounts of B.We compared the B utilization capacity and B absorption and distribution between different rootstocks,investigated the changes in the composition and structure of cell wall materials after B deprived by Solid-state 13 C Nuclear Magnetic Resonance(13C-SSNMR)and Fourier-transform infrared spectroscopy(FTIR),studied the effects of B deficiency on the microstructure in leaf and root from citrange rootstock by the method of SEM,TEM and tissue paraffin section and analyzed the changes in metabolic profiling in trifoliate orange rootstock seedlings leaf and root under B limitation using metabolomics method based on GC-MS.The major results were concluded as following:(1)Differences in plant growth and B utilization capacity between citrange and trifoliate orange rootstock under B deficiencyRoot growth and development of citrange and trifoliate orange rootstock seedlings was evidently inhibited.B-deficiency significantly decreased root parameters,and the trifoliate orange was affected more than the citrange.Under B deficiency,trifoliate orange had the greater decreased degree of photosynthetic pigment content than citrange,furthermore,B deficiency significantly reduced the net photosynthetic rate of trifoliate orange,but no significant influence on citrange.Trifoliate orange seedlings contained higher B concentration and B accumulation than citrange under control and B-deficiency treatment;the ratio of protoplast-B concentration to the total B in various parts of trifoliate orange plants was much higher than that of citrange;boron utilization index in citrange was 29.13% greater than trifoliate orange.These results suggested that the demand for B in trifoliate orange was higher than citrange and citrange had less B but produced relatively more dry mass.B-deficiency significantly increased the ratio of B concentration in cell wall to the total B of leaves and roots in both types of rootstocks,192.08% and 131.03% from citrange are bigger than 166.67% and 112.70% from trifoliate orange.These results demonstrated that citrange have greater B distribution capacity in cell walls than trifoliate orange.Therefore,trifoliate orange is more sensitive to B deficiency than citrange.(2)Differences in B absorption and distribution between citrange and trifoliate orange rootstock under B deficiencyThe results of q RT-PCR analysis shown that the overexpression of B-related genes was observed more in root and old stem of citrange,while detected more in leaf and stem of trifoliate orange.Significant up-regulation of gene Ci NIP5;1 were found both in these two types of rootstock and B was accumulated in roots,suggested more B could be uptaked by roots under B deprivation.But the Ci NIP5;1 overexpression level from citrange was higher than trifoliate orange,suggested B absorption ability in citrange was much greater than trifoliate orange under B deprivation.B-deficiency clearly up-regulated the expression level of B transporter gene Cm BOR1 in root of citrange,modulated B transport and distribution in shoot of citrange plants due to its role in loading B into the xylem,but no effect on trifoliate orange.However,the expression level of genes NIP6;1,Cm BOR1,Ci NIP5;1 and TIP4;1 from leaf/stem in trifoliate orange were significantly increased due to B deprivation,involved in limited B transport and distribution in overground part of trifoliate orange plant.(3)Effect of B deficiency on membrane permeability and activity of antioxidant enzyme of leaves and roots in citrange rootstock seedlingsBoron deficiency inhibited the growth and development of both shoots and roots in citrange rootstock seedlings,and the roots were affected more than shoots.B concentration and B accumulation from various parts of citrange were evidently reduced due to B deficiency.B deficiency clearly increased MDA content of leaves and roots,55.21% for roots was higher 23.85% for leaves,suggested membrane permeability of roots was damaged more seriously than that of leaves.After B deprived,SOD activity in roots was significantly increased;CAT activity in leaves and roots was clearly decreased,but the activity of APX and POD were significantly increased.The biggest amplitude of variation was POD activity in roots,increased by 117%,shown that POD could involve in the other metabolism process in roots.(4)Effect of B deficiency on the microstructure in leaves and roots and fine-root growth of citrange rootstock seedlingsFrom the paraffin section micrographs of leaves,we observed that B-deficiency treatment led to a disordered arrangement of palisade tissue cells,obviously increased the number of spongy tissue cell,and inhibited the development of the vascular bundle.After short-term boron deficiency,changes in mesophyll tissue and vascular bundle structure degree were not enough to destroy the leaf epidermis,so the obvious B-deficiency symptoms wasn’t observed in the leaves of citrange.The results from transmission electron microscopy(TEM)shown that B-deficiency led to the accumulation of starch grains in the chloroplast,obviously thickened cell walls and changed cell morphology of leaves.Root parameters of its fine root were decreased more greater than coarse roots,suggested the growth and development of fine roots were mainly inhibited by B-deficiency.The proportion of total root length and root tips from coarse roots was significantly increased,showed that B deficiency caused the root tips become obviously thicker and browner.The paraffin section micrographs of roots showed that the epidermal cells of root tips were arranged loosely,and some part of them were broken down and fallen off,moreover the parenchyma cells in cortex were arranged at random and freely presented in various sizes;besides,B deficiency also generated undeveloped structure of vascular tissue.Using scanning electron microscopy(SEM)and TEM,we observed that B-deficiency destroyed the integrity of cell structure;inhibited cellular content synthesis and vascular bundles development;led to xylem vessel smaller and blocked and phloem sieve-tube disappeared;obviously made the cell wall thickening.These results could explain why B-deprivation caused root color darker and apical enlargement.(5)Effect of B deficiency on chemical composition and structure of root cell wall of citrange rootstock seedlingsThe result of this study showed that the growth and development of new roots in citrange rootstock seedlings were evidently inhibited by B deficiency.Boron deficiency significantly increased the content of CWM per unit of root fresh weight and the ratio of B concentration in cell wall to the total B in roots.In the absence of B supply,considerable differences were observed in CWM FTIR spectra in three characteristic absorption regions: 3452,1641 and 1400cm-1.The band at 3429 cm-1 in the plant cell wall of normal B condition was shifted to 3442 cm-1 when B was deprived,suggesting that the mode of hydrogen bonding was changed by B deficiency.Boron deficiency clearly decreased the peak height of carboxylic ester band around 1741 cm-1,but increased that of COO-stretching band around 1400 cm-1,suggesting that the relative amount and degree of esterification of carboxylic groups was decreased and pectin content(covalently bound pectin)and structure was altered.Furthermore,the spectra also indicated that B deficiency decreased content of root cell wall protein.(6)Effect of B deficiency on metabolite profile and metabolic pathway of leaves and roots in trifoliate orange rootstockWe concentrated on the changes in metabolic profiles of trifoliate orange leaves and roots under B deficiency by gas chromatography-mass spectrometry(GC-MS)-based metabolomics.The distinct patterns of alterations in metabolites observed in leaves and roots due to B deficiency suggest the presence of specific organ responses to B starvation.Among the metabolites with greater fold changes in concentration due to B-deficiency,the metabolites with up-accumulation in leaves were mainly sucrose,melibiose and ribose,while the metabolites with down-accumulation in leaves were galactonate,sedoheptulose,4-OH-Ci A,ferulate,palmitate and trans-sinapinic acid.For roots,however,all the metabolites of more than 1-fold changes showed significantly enhanced levels due to B deficiency,including asparagine,threonine,tryptophan,ferulate and trans-sinapinic acid.The accumulation of soluble sugars was occurred in leaves,which may be attributed to down-regulated pentose phosphate pathway(PPP)and amino acid biosynthesis under B deficiency,while the amount of most amino acids in roots was increased,indicating that the effects of B deficiency on amino acids metabolism in trifoliate orange may be a consequence of disruptions in root tissues and decreased protein biosynthesis.Several important products of shikimate pathway were also significantly affected by B deficiency,which may be related to abnormal growth of roots induced by B deficiency.