| Biological herbicide,an efficient and weed-specific biological preparation,elicits tissues and metabolites of microorganisms,plants and animals in nature to carry out industrial production.Using natural products to develop biogenic herbicides has become one of the important directions of future bioherbicide research and development.Griseofulvin,a chlorine-containing secondary metabolite isolated from Penicillium griseofulvin,is widely used in clinical medicine.In animal cells,griseofulvin affects the assembly and depolymerization of microtubules and may target microtubes-associated proteins MAPs and γ-tubulin.However,there are few studies on griseofulvin in plant,especially in weed management.Our previous studies have found that griseofulvin can inhibit the root elongation growth of Arabidopsis thaliana and induce the enlargement of root tip elongation zone,which is similar to the commercial herbicide trifluralin.Trifluralin is a root and stem inhibiting herbicide.Trifluralin interferes with plant cell division by binding with α and ?-tubulin,causes the expansion of root apical meristems and finally inhibit root elongation and growth.Further studies have shown that griseofulvin can induce the mass production of Reactive Oxygen Species(ROS)in root cells,leading to chromatin condensation and ultimately cell death.However,the exact target and mechanism of griseofulvin inhibiting root growth in plants remain unclear.This paper revealing the target and mechanism of griseofulvin to inhibition of root growth by analyzing the relationships between treatment of griseofulvin and trifluralin,microtubule states,ROS production,cell death and root growth together with microscopic detection and transcriptome sequencing analysis,so as to find out new herbicide action targets and provide theoretical basis for the development of new griseofulvin-based bio-herbicides in the future.Firstly,cell death detection of A.thaliana roots was conducted by TBD staining after treatment with griseofulvin and trifluralin for 5 days.The results showed that there were rare dead cells found in root tip at the concentration of 10μM,and the number of dead cells increased with elevated concentration.However,at the concentration of 40μM,severe cell death occurred in transition zone and elongation zone of griseofulvin,while trifluralin mainly affect meristematic zone.To precisely analyze how griseofulvin and trifluralin affect different zones in root tips,cell activity and morphology of root tip cells were detected by PI staining.After 9 h treatment with 40μM griseofulvin and trifluralin,cells of root tip showed pronounced changes.Griseofulvin mainly leads to cell membrane rupture in elongation zone while trifluralin mainly leads to that in meristem zone.After 24 h treatment,the cell enlargement in elongation zone and cell rupture in the meristems zone were detected under griseofulvin treatment,while severe cell rupture in all zones and cell enlargement in meristems zone were detected in the case of trifluralin.In addition,the results of FDA staining also showed that the cell activity of A.thaliana root was significantly reduced after 40μM trifluralin treatment for 1 to 3 days,and the cell activity of all zones of the root was almost completely lost on the third day,which was the same as previous results in griseofulvin.Cell vitality can be partially restored after pretreatment with ROS scavengers,such as DMTU,DPI,SOD and NAC,which suggested that griseofulvin-and trifluralin-induced cell death in root tip was directly related to ROS production.The results of DAB and NBT staining showed that ROS accumulation in roots was detected after 3 h treatment with 40μM griseofulvin and trifluralin,and the ROS production level reached the peak at 9 h.The transitional region is the main area of ROS accumulation after griseofulvin treatment,while the ROS induced by trifluralin are mainly produced in the meristem.This result is consistent with the earliest site of root cell death and expansion.By detection with fluorescence microscope combined with fluorescein dichlorofluorescein H2DCF-DA probe,it was found that ROS were produced in roots after treated with 40μM griseofulvin and trifluralin for 0.5 h.It follows that ROS production is an early event in the process of cell death and root growth inhibition induced by griseofulvin and trifluralin.However,what is the connection between cell death and microtubules?In order to probe the relationship between morphological structure of microtubules and root inhibition with trifluralin and griseofulvin treatment,MBD-GFP,a wild-type(Col-0)microtubule green fluorescent labeling material of A.thaliana was monitored with laser confocal microscope.The cortical microtubules in each apical region in control plants were complete in structure and normal in morphology.Cell microtubules in the meristem and transition regions were arranged horizontally,while that of elongation zone were inclined partially,that of mature zone were arranged vertically.Treated with 40 μM griseofulvin,the microtubule morphology of different root zone remained intact within 1 h;after 3 h,the cell microtubules in the transition zone and the elongation zone were depolymerized,while that of meristem zone remained normal,that of mature zone did not have significant changes;after 6 h,microtubules in transition zone and elongation zone had disintegrated,while that of meristematic zone and mature zone remained intact;after 12 h,microtubules in meristem zone began to disintegrate but cell morphology remained relatively intact,while that of transition zone and elongation zone began to show enlargement and deformation;after 24 h,microtubules of meristem,transition and elongation zones almost disintegrated completely along with some cells breaking up,while that of mature zone began to disintegrate.It can be seen that the effect of griseofulvin on root microtubules first occurred in the transitional elongation zone,which is consistent with the analysis of ROS production and cell death.Rather,microtubule depolymerization is a relatively late event compared to ROS production.This indicates that by binding to original target,griseofulvin induces ROS production,microtubule depolymerization,cell death,as well as the enlargement of root elongation zone and eventually induces root growth inhibition.On the other hand,with 40μM trifluralin treatment,microtubules of cells in each zone had severe disintegration within 1 h,but the cell morphology remained intact.The cells in meristem zone and transition zone began to deform and disintegrate at 6h,while that of elongation zone and the mature zone began to deform and disintegrate at 12 h.For further analysis of the initial site of trifluralin on root tip,microtubules and cell morphology in the root zone were measured after treated with 20 μM trifluralin.Treated with low concentration trifluralin for 10 min,the microtubules in the root zones did not change significantly.After 20 min,the microtubules in the meristematic zone began to disintegrate,while that of other zones were normal.After 1 h,microtubules of most cells in meristem,transition and elongation zones disintegrated,while that of mature zone began to disintegrate,and the cells in meristematic zone began to break up.The results showed that trifluralin can damage microtubules very rapidly:the effect of low concentration trifluralin on root microtubules initially occurs in the meristematic zone,and high concentration trifluralin causes serious damage to the microtubules in all zones.This suggests that the effect of trifluralin on microtubules may be an early event or an independent event compared to ROS production.Trifluralin induces ROS production by binding to tubulin-binding proteins and damages its structure,or by acting with other targets,subsequently induces cell death,and ultimately leads to decreased cell viability,tissue enlargement and inhibition of root growth and development.However,why do microtubules from cells in different root regions show different sensitivities to griseofulvin and trifluralin treatment?To solve this question,we performed transcriptome sequencing analysis of the plants at the time that do not have changes in microtubule morphology of root tip cells after griseofulvin and trifluralin treatment.Through transcriptome analysis of A.thaliana roots treated with 40 μM griseofulvin for 1 h and 20 μM trifluralin for 10 min,there were 921 and 560 genes respectively that differentially expressed in the early stage(microtubules have not yet occurred).Among these genes,115 genes were common in both treatments.Combined with Mapman,GO enrichment and KEGG pathway analysis,we found that griseofulvin mainly affects the expression of auxin-,ethylene-,cytokinin-and gibberellin-related genes,containing 14,9,4 and 3 related genes,respectively.Thereinto,auxin-and ethylene-related genes had the most significant differences with 9 and 6 genes up-regulated respectively.The auxin-related aluminum-inducible protein gene Alin(AT4G27450),SAUR-like auxin-reactive protein family gene(AT2G37030),the ethylene synthase gene ACS6(AT4G11280)and the ethylene-responsive transcription factor ETR2(AT3G23150)were mostly differentially expressed.Trifluralin mainly affected auxin-related genes,there were 13 genes significantly down-regulated out of 15 differential genes,of which SAUR-like auxin-reactive protein family genes takes the major part.It is worth noting that griseofulvin and trifluralin affected completely different auxin genes.In addition,they both have significant impacts on some microtubule-associated protein genes.Griseofulvin significantly increases the expression levels of cellulose synthase CSLA10(AT1G24070)and tubulin-tyrosine ligase TTL(AT4G21920),and significantly decreases the expression of cellulose-synthase-like protein CSLB4(AT2G32540),cyclin protein CYCU1-1(AT3G21870)and tubulin protein TUBB5(AT1G20010).These five genes are involved in the assembly and dynamic stability of microtubules.Rather,trifluralin had a significant effect on the microtubule-associated protein genes TPX2(AT3G01710),MADA1(AT4G02800)and CDC20-1(AT4G33270).This indicates that except the microtubule structural proteins α and ?-tubulin,auxin-related proteins and these three microtubule-associated proteins may also be the initial targets of trifluralin.Whereas,the main action sites of griseofulvin may be auxin-and ethylene-related genes,as well as microtubule-associated cellulose synthase CSLA10,tubulin-tyrosine ligase TTL,cellulose synthase-like protein CSLB4,cell cycle protein gene CYCU1-1 and tubulin protein gene TUBB5,rather than microtubule structural protein α and ?-tubulin.Finally,based on the above results combined with previous studies,we proposed an action pattern of griseofulvin and trifluralin.Griseofulvin induces the production of auxin and ethylene in roots by affecting the expression of auxin-and ethylene-related genes,thereby induces the accumulation of ROS,cell death and tissue enlargement in the elongation zone,and finally resulting in root growth inhibition;besides,griseofulvin can also affect the expression of non-microtubule structural protein gene,so that affect the structure and dynamic stability of microtubules,leading to cell deformation,disintegration and root growth inhibition.Trifluralin can induce cell microtubule depolymerization by binding with microtubule structural proteins α and ?-tubulin,which arousing cell deformation and disintegration and root growth inhibition.At the same time,trifluralin can also reduce auxin production in roots by inhibiting the expression of auxin-related genes,then triggers accumulation of ROS in the meristematic zone,cell death,tissue enlargement,and finally results in root growth inhibition. |
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