| Salvia rosmarinus as a new aromatic shrub with high utilization value,is widely planted in many countries in the world,and its planting scale in China is also expanding.Salvia rosmarinus is an intolerant plant,and water condition is one of the main factors affecting its growth and development.The serious loss caused by waterlogging disaster has become one of the factors restricting the development of Salvia rosmarinus industry.Therefore,it is very important to explore the development of Salvia rosmarinus roots and the damage mechanism under flooding stress.In this paper,Salvia rosmarinus(Albus 2)was used as the research object,and cultivated with different concentrations of nutrient solution.By measuring the morphology,anatomical structure and physiological indexes of Salvia rosmarinus roots,the effects of soil culture and hydroponic culture on root development were explored.In addition,in order to further reveal the mechanism of Salvia rosmarinus seedlings in response to flooding stress,this study combined morphological,physiological and transcriptome analysis to deeply explore the physiological and molecular mechanisms of Salvia rosmarinus roots in response to flooding stress,providing theoretical guidance for the cultivation techniques of Salvia rosmarinus and the breeding of highly resistant varieties.The main results are as follows :(1)Root morphological characteristics and comparative analysis of soil culture and hydroponic Salvia rosmarinus.The results of root scanning showed that the root length,root volume and root surface area of Salvia rosmarinus in soil culture group increased with the increase of nutrient solution concentration,and the root development of T1 group(full concentration nutrient solution culture)was the best.The root length,root volume and root surface area of Salvia rosmarinus decreased with the increase of nutrient solution concentration,and the root development of S3group(1 / 4 nutrient solution culture)was the best.According to the observation of the anatomical structure of Salvia rosmarinus root system by paraffin section,it was found that the number of layers of outer cortex cells in hydroponic Salvia rosmarinus root system was less than that in soil culture group,and the cell arrangement was looser.There were abundant aerenchyma in cortical parenchyma cells.The diameter of endodermis and stele sheath cells decreased and arranged more closely.The differentiation of xylem and phloem cells was not obvious.The xylem of soil culture Salvia rosmarinus root system had obvious large vessels.With the increase of nutrient solution concentration,the outer cortex and cortex parenchyma cells of soil-cultured Salvia rosmarinus root system were arranged more closely,and the apical meristem area also occupied a large proportion in the root anatomical structure.The growth and development of root tip became vigorous with the increase of nutrient solution concentration.With the increase of nutrient solution concentration,the cells in the root apical meristem of the hydroponic group were more closely arranged,the number of divisions was more,and the cells in the elongation zone and mature zone were more regularly arranged.(2)Soil culture and hydroponics Salvia rosmarinus root physiological variation and comparative analysis.There were significant differences in the physiological state of Salvia rosmarinus roots under hydroponic and soil culture conditions.The content of malondialdehyde(MDA)in the roots of the soil culture group and the hydroponic group decreased with the decrease of the concentration of the nutrient solution.The activity of superoxide dismutase(SOD)in the soil culture group decreased with the decrease of the concentration of the nutrient solution.The hydroponic group showed a trend of increasing first and then decreasing.The activity of peroxidase(POD)and ascorbate peroxidase(APX)in soil culture group decreased first and then increased with the decrease of nutrient solution concentration.The activity of POD and APX in the hydroponic group showed a trend of increasing first and then decreasing.The activity of Catalase CAT in the roots of soil culture group and hydroponic group rose with the decrease of nutrient solution concentration.The content of proline(Proline Pro)and soluble protein in the roots of Salvia rosmarinus in soil culture group decreased first and then increased with the decrease of nutrient solution concentration.The content of soluble protein in hydroponic group also decreased first and then increased with the decrease of nutrient solution concentration,while proline showed a downward trend.(3)Effects of flooding stress on plant morphology and root tissue structure of Salvia rosmarinus.By observing the changes of plant morphology of Salvia rosmarinus seedlings under different flooding degrees and flooding times,it was found that during the whole flooding stress period,the plant morphology of Salvia rosmarinus under root submergence treatment remained good,and no obvious phenotypic changes occurred,indicating that the stress degree of root submergence had little effect on the morphological growth of Salvia rosmarinus plants.Under the stress condition of full root flooding,the leaves of Salvia rosmarinus will appear curling,wilting,yellowing and other waterlogging phenomena.With the prolongation of stress time,the symptoms of waterlogging will be aggravated.Water flooding caused some cells in the cortical parenchyma of Salvia rosmarinus roots to die to form cavities,and the endodermis,stele sheath and phloem were damaged to varying degrees.In the first 6 days of root submergence and full submergence,the relatively complete cell structure was maintained.The root cell structure was seriously damaged on the 9th day of root submergence,and the root system was basically dead on the 9th day of root full submergence.Therefore,with the increase of water depth and the extension of submergence time,the anatomical structure of Salvia rosmarinus roots was damaged.(4)Physiological response of Salvia rosmarinus roots to flooding stress.With the increase of flooding stress time,the MDA content of Salvia rosmarinus roots in HF(semi-submerged)group increased,the activities of SOD,POD and APX increased,and the activity of CAT increased first and then decreased.The MDA content of Salvia rosmarinus roots in TF(full flooding)group decreased,the activities of SOD,POD and CAT decreased,and the activity of APX increased first and then decreased.The Pro and soluble protein content of Salvia rosmarinus roots in HF(semi-flooded)group decreased first and then increased.The Pro content of Salvia rosmarinus roots in the TF group showed a trend of increasing first and then decreasing,and the soluble protein content showed a trend of decreasing first and then increasing.(5)Transcriptome mechanism of Salvia rosmarinus roots in response to flooding stress.The transcriptome analysis of Salvia rosmarinus roots under flooding stress was carried out,and the GO enrichment analysis of the selected DEGs showed that under the condition of root semi-flooding,DEGs were mainly enriched in biological processes such as antioxidant response,and under the condition of root full flooding,DEGs were mainly enriched in biological processes such as response to inorganic substances.The KEGG pathway enrichment analysis of the screened DEGs showed that the pathways of monoterpene biosynthesis,sesquiterpene and triterpenoid biosynthesis were significantly enriched under root submergence,and the number of differentially expressed genes was enriched in plant hormone signal transduction,phenylpropanol biosynthesis,starch and sucrose metabolism,sesquiterpene and triterpenoid biosynthesis.The pathways of steroid biosynthesis and N-glycan biosynthesis were significantly enriched under root flooding,and the number of genes enriched in amino acid biosynthesis,phenylpropanol biosynthesis and endoplasmic reticulum protein processing was higher.The antioxidant enzyme system of Salvia rosmarinus seedling roots was mainly annotated to five pathways in response to flooding stress,namely glutathione metabolism(ko00480),phenylpropanoid biosynthesis(ko00940),peroxidase(ko04146),ascorbic acid metabolism(ko00053)and tryptophan metabolism(ko00380)pathways.By analyzing the differential expression of related genes,it was found that the expression of POD-related differential genes in antioxidant enzymes was the highest.Among them,there were 58 POD-related genes in CK-6_vs_TF-6 group,of which 20 were up-regulated and 38 were down-regulated.There were 22 genes with | Log2 FC | ≥ 5,of which 6 were up-regulated and 16 were down-regulated.Therefore,POD may be the main factor for Salvia rosmarinus roots to scavenge excessive reactive oxygen species.The Pro-related genes screened from the roots of Salvia rosmarinus seedlings under the stress of root flooding were mainly annotated to three pathways : aminoacyl t RNA biosynthesis(ko00970),other types of O-glycan biosynthesis(ko00514),arginine and proline metabolism(ko00330)pathway.The analysis of differential genes related to osmotic adjustment substances showed that there were significant differences in the expression of Pro-related genes.There were 32 Pro-related genes in CK-6_vs_TF-6 group,of which 6 were up-regulated and 26 were down-regulated.There were 12 genes with | Log2 FC | ≥ 5,of which 1 was up-regulated and 11 were down-regulated.The experimental results showed that flooding stress caused differential expression of genes related to antioxidant enzymes and osmotic regulators in Salvia rosmarinus roots,which affected related physiological processes by regulating the differential expression of related pathway genes,and ultimately enhanced the adaptability to flooding environment.In summary,this study analyzed the changes of root morphology,anatomical structure and physiological indexes of Salvia rosmarinus under different concentrations of nutrient solution,and compared the differences between soil culture and hydroponic culture,and explored the optimal nutrient solution concentration suitable for soil culture and hydroponic culture.Furthermore,the changes of anatomical structure and physiological indexes of Salvia rosmarinus roots under different flooding time and flooding degree were clarified by flooding stress.Combined with transcriptome sequencing,the molecular mechanism of Salvia rosmarinus roots in response to flooding stress was preliminarily clarified,and the key genes and KEGG pathways of antioxidant enzyme system in response to flooding stress were screened out,which provided a theoretical reference for further cultivation of new lines of waterlogging-tolerant Salvia rosmarinus. |
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