Genotypic Differences In Cell Wall Mechanical Properties,Phytohormone And Reactive Oxygen Species Signals In The Responses Of Oilseed Rape To Low Boron Stresses

Brassica napus is greatly susceptible to boron(B)deficiency.B deficiency seriously reduces the seed yield of rapeseed.B resources are deficient in the main cultivation area of rapeseed in China.At present,application of B fertilizer is the main measure to address this problem in the agricultural industry.However,B rock is a non-renewable mineral resource and inappropriate application of boron may cause the B toxicity.Hence,selecting rapeseed B-efficient cultivars and mining the physiological and molecular mechanism underlying B-efficiency is theoretically and practically significant for the high quality and yield of rapeseed.The B-efficient and-inefficient rapeseed cultivars have been indentified,respectively.And the physiological mechanism and genetic basis underlying B-efficiency has been explored in the past few years.However,the mechanism involves not only the absorption,transport and distribution of B,but also the regulation of phytohormone,reactive oxygen species and calcium signal and even involves the complex interaction among plant cells,tissues and organs.We established a suspension cell system from the B-efficient genotype ‘QY10’ and B-inefficient genotype ‘W10’.Using a combination of physical,chemical and molecular biological techniques and various microscopic observation methods,we investigated the physiological mechanism underlying B efficiency from the perspective of phytohormones,pectin,reactive oxygen species and Ca2+.The main results are summarized below:1 The responses of suspension cell of B-efficient and inefficient genotypes to low-BHere,an efficient suspension cell system derived from the rapeseed hypocotyl was established and the differences of low-B responses between genotypes were studied.With the decrease in B concentration,the cell viability was significantly reduced and the deformed cells became more.Under 0.1 μM and 0.25 μM B conditions,the suspension cells of ‘W10’ showed lower viabilities and less cell total number than ‘QY10’,indicating the B-efficiency was significantly different at single cell level.The system is not only suitable for B efficiency evaluation but also for the study on rapeseed as a single cell model.2 Pectin-mediated cell wall mechanical property confers the differential low-B sensitivities between genotypesUsing the scanning electron microscope(SEM)and transmission electron microscope(TEM),a more swollen and more easily ruptured cell wall(CW)could be observed in ‘W10’ under B deficiency.The cell rupture was attributed to the weakened CW mechanical strength of ‘W10’ detected by atomic force microscopy(AFM).The mechanical strength differences between ‘QY10’ and ‘W10’ were diminished under sufficient B condition or after the removal of pectin.Further,we found ‘W10’ exhibited significantly higher pectin concentration with much more structurally unstable RG-II monomer than ‘QY10’ under B deficiency.Through DGE test and quantitative real time polymerase chain reaction(q RT-PCR),we found ‘W10’ presented obviously elevated m RNA abundances of pectin biosynthesis-related genes than ‘QY10’ under B deficiency.The great differences in pectin concentration and monomeric RG-II ratio were verified by analyzing the low-B-sensitive and low-B-tolerant rapeseed plants selected from a natural population.These results indicated that the lower pectin concentration on rapeseed cell wall is more favorable for plants to resist low-B stress.3 The spatial-and temporal-responses of phytohormone and its related genes to B-deficiencyDigital gene expression(DGE)profiling revealed that the expression of many phytohormone-related genes was different between genotypes.Here,we focused on indole-3-acetic acid(IAA),jasmonic acid(JA)and abscisic acid(ABA)in response to low-B.Our study indicated low-B reduced IAA concentration and the expression of the auxin biosynthesis and efflux genes,and increased the JA and ABA concentrations together with the expression of ABA biosynthesis and sensor genes of B.napus under long-term B deficiency.In two contrasting genotypes,the auxin concentration decreased more drastically in the B-inefficient genotype ‘W10’,while the JAs and ABA concentrations were considerably higher in this genotype.The concentration of auxin and the expression of IAA efflux genes was significantly decreased in the shoot apical and root of ‘W10’,which promoted the outgrowth of lateral branches and inhibited the root growth,respectively.The application of exogenous IAA could partly restore the B-defective phenotype in ‘W10’.Overall,our data suggested that differences in IAA between genotypes may partly account for their differences in tolerance to low-B(B-efficiency).4 Involvement of reactive oxygen species(ROS)and Ca2+ in the differential responses to low-B in rapeseed genotypesLow-B induced O2-accumulation,whose distribution was similar to the cell death regions in the plant roots.The increase in O2-production was more violent in ‘W10’ than in ‘QY10’ under both plant root and suspension cell systems and pretreatment with O2-scavenger increased the cell viabilities.The change trend of H2O2 was similar to that of O2-,whereas less significant.Pretreatment with the H2O2 scavenger did not significantly increase the cell viabilities.B deficiency caused the membrane lipid peroxidation,leading to the severe ion leakage including K+ efflux in ‘W10’.Low-B induced Ca2+ influx and pretreatment with Ca2+ channel inhibitor could significantly reduce the accumulation of ROS caused by low-B,indicating Ca2+ worked upstream of ROS.Through detecting the expression of genes encoding ROS-generating enzymes and the activities of antioxidant enzymes between genotypes,we found it was not the ability to scavenge ROS but the differences in ROS generation that determined the differential oxidative damages in rapeseed genotypes.