| As one of the most important abiotic stresses in plants,drought seriously restricts the growth,yield and quality of crops.Cotton serves as an important economic crop worldwide and it reveals relatively high tolerance to abiotic stress,while drought still poses a great threat to cotton production.As the organ that directly contacts the soil,the root system first perceives the water deficit.Micro-roots(namely fine roots and root hair)are the most sensitive and active parts of root system,serving as an essential organ for plants to obtain water.However,research on the morphology underlying micro-roots responses to soil drought has not been well understood in field crops.Therefore,in this study,two kinds of pot experiments(self-assembled in situ roots observation method and traditional pot culture method)have been carried out in an artificial climate chamber by taking upland cotton(Gossypium hirsutum L.)cultivar "Guoxin No.9" as an experimental material.(70±5)%and(40±5)%relative soil water content(RSWC)treatments were imposed on control(CK)and drought stress(DS)groups,respectively.By investigating the morphological and physiological characteristics of cotton,combined with differential proteomic analysis,the effects of drought stress on the development of cotton above-ground,the morphological characteristics of micro-roots and the possible regulatory mechanism were explored,the classifications and functions of micro-roots drought response proteins were analyzed.The main results are as follows:1.The self-assembled "cotton culture and in situ observation of the root system"integrated device installed in high resolution scanner can meet the demands of healthy growth of cotton and non-destructive in situ observation on the root system simultaneously,thus morphological characteristics of fine roots and root hair can be clearly observed.2.Drought stress inhibited the development of cotton above-ground.Plant height and stem diameter of DS treatment decreased significantly on 15 DAD(Days After Drought Treatment),compared with CK,and the leaf area decreased on 30 DAD,the relative chlorophyll content(SPAD value)of main stem functional leaves was decreased,the relative water content of leaves declined,and lower leaves turned yellow,became wilted,ultimately falling off.3.Drought stress inhibited the photosynthetic performance of main stem functional leaves of cotton and improved the water use efficiency.Compared with the CK group,the photosynthetic gas exchange parameters,including net photosynthetic rate,transpiration rate,stomatal conductance and intercellular CO2 concentration of the DS treatment were decreased on 30 DAD and reached a significant level of difference(P<0.01);the instantaneous water use efficiency and intrinsic water use efficiency of leaves both significantly increased on 30 DAD and 45 DAD(P<0.01),but decreased to the same level as the CK treatment on 60 DAD.4.Facing with soil drought,the root elongated,and the diameter decreased.In the high-resolution root images obtained through scanners,it was found that in the 9th week of drought treatment,the root length density demonstrated a dramatic increase of 17.08%,compared with CK(P<0.01),the average diameter significant decreased by 16.03%,and the change of surface area density showed no regularity.And the results of total root length,total root surface area and total average root diameter obtained by the traditional pot culture method were similar to the above,which indicated that the response trends of partial root system and total root system to drought stress were well consistent.5.Drought stress accelerated the death of fine roots and root hairs,promoted the elongation of root hairs.Fine root lifespan and its diameter were positive correlated(R2=0.6933),the larger the diameter is,the longer the fine root lives,and this relationship did not change under drought treatment(R2=0.7342),but the lifespan of fine roots decreased.Drought stress also accelerated the death of root hairs.Compared with fine roots,root hairs have a shorter lifespan,but they are the first part to respond to drought.The lifespan of root hairs varies in different growth stages,and the lifespan in the vegetative growth stage was longer than that in the reproductive stage.Under CK,their lifespan were 21 d and 14 d,respectively,and DS treatment was shorened to 19 d and 13 d,respectively.In the second week of drought treatment,the root hair length of DS was significantly higher than that of the CK treatment(P<0.01).With the extension of stress time,the differences of the root hairs length between the two treatments increased gradually,and the peak showed an increase of 88.01%compared with CK.6.Drought stress enhanced the activities of antioxidant enzymes,increased the degree of membrane lipid peroxidation and caused the accumulation of osmotic adjustment substances.Antioxidant enzyme activities and the contents of non-enzyme antioxidants showed significant increase on 15 DAD,compared with CK.At the same time,MDA content in the DS group was dramatically higher than that in the CK(P<0.01),and H2O2 content was significantly higher on 30 DAD(P<0.05).The contents of proline and soluble sugar under DS treatment were obviously higher than those under CK treatment on 30 DAD.7.Based on the technique of tandem mass tag,differential proteomics analysis has been performed on cotton fine roots(CK and DS treatments)on 0 DAD,30 DAD and 45 DAD.Results showed that a total of 11,628 identified proteins,of which 10,344 contained quantitative information.On the basis of fine roots morphology,physiological and biochemical results,samples with 30 DAD and 45 DAD were focused observed,and the value showing more than 1.3-fold or less than 0.77-fold changes were adopted to assess up-regulated and down-regulated,respectively.223 and 1,273 differential expression proteins(DEPs)were obtained from the group of "DS30 vs CK30" and that of "DS45 vs CK45",respectively.8.GO annotation divided the DEPs into three categories,consisting of biological process(mainly of metabolic process,cellular process,single-organism process),cell component(mainly of cell,organelle,cell membrane)and molecular function(mainly of binding,catalytic activity).Through the KEGG pathway enrichment analysis,it can be found that in the comparison group of“DS30 vs CK30",up-regulated DEPs were mainly enriched in "Cutin,suberine and wax biosynthesis","Glycosphingolipid biosynthesis","Galactose metabolism","Pentose and glucuronate conversion" pathways.Down-regulated DEPs were enriched in "Monoterpenoid biosynthesis","Carbon fixation" and "Oxidative phosphorylation" pathways.In the comparison group of "DS45 vs CK45",up-regulated DEPs were mainly enriched in pathways such as "Galactose metabolism","Arginine and proline metabolism","Phagosome","Starch and sucrose metabolism" and "Amino sugar and nucleotiwokande sugar metabolism".Down-regulated DEPs were enriched in pathways such as "Biosynthesis of secondary metabolites",“Fatty acid metabolism","Fatty acid biosynthesis","Terpenoid backbone biosynthesis" and "Carotenoid biosynthesis".The enriched KEGG pathways and some important DEPs related to stress response were further divided into five categories,including carbohydrate and energy metabolism,stress and defense responses,plant hormone metabolism,lipid metabolism,amino acid metabolism,and secondary metabolism.Extensive descriptions and studies on individual proteins have been implemented to explore the DEPs with high research value in response to drought stress.In view of the above results,the phenotypic differences between cotton above-ground and the micro-roots system in response to drought stress,and the protein level difference between fine roots in response to drought can be preliminarily revealed.This will provide a basis for further research on the drought resistance mechanism of cotton,provide new ideas for crop drought stress regulation and breed drought resistant varieties,and provide necessary theoretical guidance for the optimization and improvement of root traits. |
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