Variation In Rhizosphere Features And Their Effects On The Accumulation Of Ciprofloxacin-(CIP) In High-and Low-CIP Accumulation Cultivars Of Chinese Flowering Cabbage (Brassica Parachinensis L.)

China is the largest producer and user of antibiotics in the world.Antibiotics were universal detected in soil and surface water,which directly affected the quality safety of agricultural products and caused potential risk to human health.A novel alternative strategy of screening for or breeding low-pollution accumulating crop cultivars(or pollution-safety crop cultivars)has been proposed to ensure the safety of agricultural products in this now universal antibiotics contamination of agricultural soils.Focused on the important vegetable-Chinese flowering cabbage(Brassica parachinensis L.)in southern area of our country,this paper probes into the variation in Ciprofloxacin(CIP),widely used in humans and animals,accumulation among cultivars and screening for low or high-CIP accumulation cultivars.By comparing the differences of uptake actions(including uptake pathways and assimilation kinetics)and physiological characteristics(including morphologyand anatomy,subcellular distributions,electrophysiology,etc.)and rhizosphere effects between low-and high-CIP accumulators,this study proposes to reveal the formation mechanism of high-and low-CIP accumulation cultivar.The main results are as follows:(1)Difference of uptake pathways and assimilation kinetics between low-and high-CIP accumulation cultivarsInvestigated the root uptake pathways and its factors by experiment,including experiments of bare-root,the experiment of avoided lightand with half of roots grown in clean solution and the other half in spiked solution,which indicated passive and active root uptake occurred in uptake of CIP by root.The main root pathways of Chinese flowering cabbage was active uptake.Moreover,active root uptake in SiJiu was 1.6 times more than that of CuTai,which may play an important role in the characteristic formation of high-CIP accumulation in roots.The effects of root adsorption and phloem on the root uptake were very small,but the transpiration significantly promoted the root uptake of CIP by Chinese flowering cabbage,which was the key factor on the uptake process of high-CIP accumulation.Furthermore,the experience of assimilation kinetics showed that the SiJiu had a higher ability than CuTai in the uptake rate of CIP and carrier affinity(Km),which enhanced root uptake and transportation of CIP by SiJiu.In addition,the capacity of metabolism of CIP was significantly stronger than that of CuTai,it may be the mian reason of the high-CIP accumulation cultivar having better adapt under the stress of CIP.(2)Difference of morphology and anatomybetween low and high-CIP accumulation cultivarsObserved the root of morphology,cell structure and ultra-structure by experience of roots scan,red solid green dyeing and transmission electron microscope,the significantly greater decrease in biomass and more severe toxicity to cell ultrastructures of roots was showed in CuTai compared to those in Si Jiu.By comparison to the growth inhibition of CIP in roots.The root surface area and root volume significantly increased in SiJiu,while root diameter significantly decreased compared to CuTai,which wass conducive to the uptake of CIP by SiJiu.The more numbers and bolder of pipe in transfusion tissue of SiJiu in CIP enhanced the uptake and transport of CIP by roots to shoots.Compared to SiJiu,CIP significantly inhibited the xylem area in CuTai,which indicated that the lower translocation of CIP from root to shoot was observed in CuTai,leading to the lower uptake and accumulation of CIP in CuTai.The results showed that the change of root tissue structure was the key factor on the transpiration and phloem for the transport difference of CIP by Chinese flowering cabbage.Moreover,the root ultrastructural study showed obvious differences in physiological change of roots between two cultivars.Exposed to CIP,the damage to the root tip cells in SiJiu was lower than CuTai,thus the intact cell structure and the normal mitochondrial function were observed even under high CIP stress,which ensured that the long-term continuous uptake and accumulation of CIP in high-CIP accumulation cultivar.The damage of mitochondria and cytoplasm in root tip cells of CuTai were higher,and the damage of mitochondrial dysfunction could lead to the lack of energy in active root uptake of CIP,which was the crucial reasons to the characteristic formation of high-CIP accumulation cultivar.Overall,the root tip cells of low-CIP accumulator was less tolerant to CIP stress than that of high-CIP accumulator and thus was more vulnerable to cell ultrastructure damage.To protect its roots from CIP toxicity,low-CIP accumulator tried to reduce CIP transported to and accumulation in roots by changing its physiological structure,which was a kind of resistance to CIP stress by avoidance.(3)Differences of subcellular distributions and the mechanism in response to CIP stress between the low-and high-CIP accumulatorBy comparing the concentrations of CIP in subcellular distributions of the two cultivars using differential centrifugation,CIP in roots of both cultivars were mainly observed in the organelle and cell wall,followed by the soluble ingredient of the cell.Under low CIP stress,the concentration of CIP in the cell wall of roots in low-CIP accumulator(CuTai)was obviously higher than that of roots in high-CIP accumulator(SiJiu),which illustrated CIP was easily detained by cell wall of roots of CuTai,limiting the transport process of CIP to inside of cell and eventually allowed less CIP to uptake and transport by CuTai.Corresponding,under high CIP stress,the concentrations of CIP were decreased in cell wall of root but increased in organelle and vacuole.Interesting,the concentration of CIP in vacuole of Cutai was higher than SiJiu under low-CIP stress,but the opposite result was showed under high-CIP stress,indicating vaculoar compartmentalization might play an important role in CIP tolerance and detoxification in the root tip cells of high-CIP accumulator.Therefore,high-CIP accumulator was tolerant to high CIP accumulation in the roots due to its strong metabolism capacity,detoxification,which was a kind of resistance to CIP stress by tolerance.In the oil and red dyeing experiment,the distribution characteristics of lipids were similar to the subcellular distributions of CIP in roots,which indicated lipids may be correlated with the subcellular distributions of CIP in roots,but more research were needed for the distribution relationship between lipids-carbohydrates and CIP in Chinese flowering cabbage.The translocation and accumulation of CIP in two cultivars roots could be observed directly with single-photon laserconfocal scanning microscopy(PLCSM).The results showed that there were active and passive root uptake of CIP in two cultivars,and the concentrations of CIP in cell wall and cytoplasm of SiJiu were significantly higher than that of CuTai by the fluorescence intensity of CIP.(4)Study of the differences of cell electrophysiology of two root tip cells between the low-and high-CIP accumulatorThe Study of the differences of plasma membrane potential between two root tip cells under CIP stress carred out with microelectrodes and non-incasivemicro-test technique(NMT).The depolarization of membrane potential in root tip cells of two cultivars was obviously occurred under CIP stress,and strong depolarization of membrane potential was in root of SiJiu.The driving force of transmembrane movement depend on depolarization of membrane potential,which illustrated that SiJiu have a higher capability of uptake and accumulation of CIP in root than that of Cu Tai.Monitored by H+ influx in root tip cells of the two cultivars,the concentration of CIP in SiJiu was independent of H+ influx,but H+ influx increased alone in high-CIP stress,which speculated that the potential difference was the main driving force of uptake of CIP by the root in SiJiu.The depolarization of membrane potential increased with uptake of CIP by the root in CuTai,as did H+ influx,which indicated that the uptake process of CIP by the root in CuTai could activate some protein cotransported with H+.Therefore,it presumed that CIP may be cotransported with H+ into root tip cell of Cutai,and H+ influx was the main driving force of CIP into the root of CuTai,even directly caused cell toxicity in low-CIP accumulation cultivars.(5)Study the differences of rhizosphere effect between the low-and high-CIP accumulation cultivarsThe relationship between environmental factors,microbes and the reduction of CIP in the rhizosphere millimeter microdomains was studied by aspecial-designed rhizobox experiment.The results showed that the degradation rate of CIP in soil with cultivation of SiJiu was higher than that of CuTai,and the degradation rate of CIP was the highest in the soil near the root zone,especially in 2-4 mm.Moreover,the species richness and diversity of the soils in high-CIP accumulation cultivars were higher than those of low-CIP accumulation cultivars by the analysis of ? and ? diversity,especially the microbial diversity and abundance in root zone and near the root zone,which may be the reason for the high degradation rate of CIP in the soil with high-CIP accumulation cultivars.The correlation analysis showed that the microbial diversity in the soil promoted the reduction of CIP in the soil under the low-CIP stress,while the microbial diversity in the soil inhibited the CIP reduction in the soils near the root zone and far root zone under high-CIP stress,and the inhibition of CIP reduction was stronger in the soil near the root zone with cultivation of SiJiu.Moreover,the concertration of DOM in the root zone inhibited the microbial diversity in the soil with cultivation of CuTai and eventually the lower concentration of CIP in the soil was observed.