Analysis Of Cell Characteristics,Non-target Metabonomics And Target Metabonomics In Roots Of Trifoliate Orange Rootstock Under Boron Deficiency

Boron?B?is an essential microelement for prime growth and development of citrus.However,the extremely low B availability in soils has been a major cause for loss of productivity and quality of citrus.Rootstock provides root for citrus and its growth and development directly determine the absorption ability to nutrient and adaptation ability to adversity stress of citrus.Trifoliate orange[Poncirus trifoliata?L.?Raf.]is a well-known main citrus rootstock in China and is very sensitive to B deficiency.Root is the most sensitive organ to B deficiency that results in the rapid inhibition of growth with a significantly enlarged root tip.Thus,thorough investigation on the inner link between B-deficiency symptoms and the changes on cell structure,cell wall characteristics and metabolism in root of trifoliate orange rootstock contributes to understand the physiological functions of B in citrus rootstocks and enrich the scientific theory of B nutrition.On the other hand,the study on the effects of B resupply on metabolites and metabolic pathway in B-starved trifoliate orange roots provides a theoretical basis for scientific and rational fertilization of citrus rootstock.In the present study,young plants of trifoliate orange rootstock were selected to grown in a nutrient solution with varying concentrations of B.Paraffin sectioning,transmission electron microscope?TEM?,fluorescence microscope?FM?,Fourier-transform infrared spectroscopy?FTIR?,¹³C Nuclear Magnetic Resonance(¹³C-NMR),vital staining,non-target metabolomics?GC-TOF-MS?and target metabolomics?UHPLC-MS/MS?were comprehensive used to revealed the alterations in pectin characteristics,the architecture of cell wall components,the anatomic structure,cell viability,ROS accumulation and secondary metabolites in the shikimate pathway under B starvation,and analyzed the metabolism changes induced by B deficiency and the reversibility of these changes produced by B resupply in trifoliate orange roots.The major results were concluded as following:1 Boron-deficiency-induced changes in the microstructure and cell activity characteristics of trifoliate orange root tips.Boron deficiency significant inhibited the growth of trifoliate orange seedlings,especially of roots with hindered lateral roots and swollen root tips.The results of vital staining with FDA and PI showed the red nuclei stained by PI,destroyed cell structure and weakened cell viability in B-deficient trifoliate orange root tips,while clear green patches and intact cells in control root tips.The extensively accumulated MDA in B-deficient roots also suggested the enhanced amount of membrane lipid peroxidation.In addition,B deprivation caused damages on the anatomic structure and ultra-structure of roots with random arrangement of epidermal cells,nuclear degradation of some phloem cells,thickened cell wall,vacuolar rupture and weakened adhesion between cells.In the meantime,the vital staining with fluorescence probes and chemical determination of ROS comprehensive reflected that B starvation induced accumulation of hydrogen peroxide?H₂O₂?and superoxide radicals(O₂^·-)in root tips,and although the activities of the antioxidant enzymes-superoxide dismutase?SOD?,guaiacol peroxidase?POD?and catalase?CAT?were enhanced,the superfluous ROS was not completely removed,suggesting the oxidative stress on roots under B deficiency.2 Boron-deficiency-induced changes in pectin composition and architecture of components in cell walls of trifoliate orange roots.The length of B-starved roots and shoots decreased 64.86%and 46.35%,respectively,and the dry mass accumulation of roots,stems,and leaves were also remarkably reduced by B deprivation after being treated in B-deficient solution for 9weeks.Additionally,B deficiency both decreased the B concentration in roots and cell walls,while notably increased the proportion of cell wall B in root B,indicating that B in roots was assigned preferentially to cell walls under B starvation.The results of this present study also showed thickened cell walls,more cell wall materials and destroyed structure of root cells under B deficiency.The cell wall thickness measured by Photoshop CS6 of B-deprived root tips was 1.82 folds as thick as that of the control.What is more,FTIR and ¹³C-NMR spectra revealed that B deprivation affected the architecture of chemical composition and the organic carbon structure of root cell walls.B deficiency influenced protein structure and the hydrogen bond between protein and carbohydrates?cellulose,hemicellulose?,and promoted accumulation of cellulose,amino acids,wax,lignin and phenolic compounds in cell walls.It should be noted that the uronic acid of Na₂CO₃-soluble pectin in the cell wall of roots was decreased by 34.09%under B deprivation compared with control,and KDO content of CDTA-soluble pectin and Na₂CO₃-soluble pectin were decreased by21.59 and 31.92%,respectively.Meanwhile,B deficiency increased the DM of CDTA-soluble pectin in roots.The results suggested that the swelling and weakened structural integrity of cell walls,which induced by alteration on the network of pectin and cell wall components and structure in B-deficient roots,could be a major cause of occurrence of the rapid interruption of growth and significantly enlarged root tips in trifoliate orange roots under B-insufficient condition.3 The non-target metabolomics and target metabolomics analysis of trifoliate orange roots under boron deficiency.The trifoliate orange seedlings grown in control and B-deficient conditions vary greatly in the relative concentrations of metabolites in roots.In present study,40metabolites were putatively identified at the similarity level of over 70%and chosen for further analysis.Boron deficiency down-regulated?-aminobutyric acid?GABA?,?-alanine,alanine,saccharic acid,?-ketoglutarate,quinic acid,palmitic acid,D-glyceric acid,fumaric acid and oxalic acid,while up-regulated 30 metabolites including 10 main amino acids,8 sugars,6 organic acids,3 fatty acids and 3 others.The changes induced by B starvation further affected the metabolic profiles and revealed the inhibition on sugar usage and protein biosynthesis and altered the tricarboxylic acid?TCA?cycle flux modes in trifoliate orange roots.Additionally,B deficiency promoted the process of the shikimate pathway in trifoliate orange roots.The increased phenylalanine?Phe?,tyrosine?Tyr?,in roots facilitated the biosynthesis of salicylic acid,caffeic acid and ferulic acid,and then caused a accumulation of lignin in roots.On the other hand,the biosynthesis of 3-indoleacetic acid?IAA?by tryptophan?Trp?was inhibited by B deprivation.In conclusions,changes in the important metabolites induced by B deficiency caused disturbance of the central metabolism in roots,might resulting in the abnormal morphological and structural changes in root tips of trifoliate orange seedlings.4 Metabolic responses in roots of trifoliate orange seedlings as induced by boron resupply.Boron resupply have a largely reversal of the B-starvation-induced changes in metabolism in roots and there was a difference in the recovery cycle between metabolites.Proline?Pro?,linoleic acid,6-deoxy-D-glucose and myo-inositol were quickly restored to normal levels after 1 week of B resupply,indicating they were more sensitive to B supply than others;After 2 weeks of B resupply,the galactonic acid,stearic acid and aconitic acid reached to the control levels,followed by serine?Ser?,gluconic acid,lactose,maleamate,oxalic acid,phytol and linolenic acid after 3weeks of B resupply;other metabolites showed a recovery tendency but may require a longer time of period of B resupply to achieve a full recovery,concluding that although B resupply effectively restored changes of some metabolites caused by B deficiency,the response cycles of different metabolites are quite different.Furthermore,with increasing B resupply time,lateral root development and biomass accumulation of roots were promoted significantly.The results indicated that B resupply can alleviate the B-deficiency-induced metabolic disorders and inhibition on growth of trifoliate orange roots to a large extent,but it is not sufficient for the full recovery of all changes.