Study On The Response Mechanism Of Puccinellia Tenuiflora Roots To Alkali Stress

Around 46%of saline-alkali soils worldwide contain alkaline salts such as Na HCO₃and/or Na₂CO₃,causing soil alkalization which has led to serious environmental problems in many regions around the world,especially in Northeast China.Puccinellia tenuiflora is an extremely alkali-resistant salt-tolerant forage grass.Due to its strong alkali resistance and high feeding value,P.tenuiflora has been widely used to restore and utilize the alkalized soil in Northeast China.Research on the alkali resistance physiology and molecular mechanism of P.tenuiflora not only enriches the theoretical framework of plant stress biology,but also provides certain support for the development and utilization of alkalized land.Currently,the salt tolerance mechanism of plants has been extensively studied,but research on the alkali resistance mechanism of plants is relatively scarce.Previous studies have shown that Na⁺detoxification,antioxidation,and root secretion are the primary mechanisms of plants to resist alkali stress.In this study,P.tenuiflora seedlings were treated with alkali stress（Na₂CO₃:Na HCO₃=1:1,100 m M）,and the content of different ions in the roots was measured.Confocal laser scanning microscopy,paraffin-embedded tissue section techniques,and other methods were used to detect the distribution of Na⁺,Ca²⁺,ROS,flavonols and other ions or signal molecules in different types of root cells.Transcriptomic sequencing was used to analyze the changes in the expression of Na⁺detoxification and antioxidation-related genes in the roots,and thus the Na⁺detoxification and antioxidation mechanisms of P.tenuiflora roots under alkali stress were explored:（1）Glycolysis,the pentose phosphate pathway,and fatty acidβ-oxidation are key steps in plant respiration.6-phosphofructokinase（PFK）and pyruvate kinase（PK）are limiting enzymes in the glycolytic pathway,glucose-6-phosphate dehydrogenase（G6PD）is the limiting enzyme in the pentose phosphate pathway,and 3-ketoacyl Co A thiolase（3-KAT）is a crucial enzyme in fatty acidβ-oxidation.Transcriptomic analysis showed that the expression of PFK,PK,G6PD,and 3-KAT in P.tenuiflora roots was up-regulated after alkali stress treatment,indicating that alkali stress may enhance glycolysis,the pentose phosphate pathway,and fatty acidβ-oxidation in P.tenuiflora roots.Enhanced respiration will further provide materials and energy for the many physiological responses and secretion regulation processes that P.tenuiflora roots undergo in response to alkali stress.（2）Alkali stress can lead to Ca²⁺precipitation around the roots,causing a sharp decrease in the available Ca²⁺content.However,after alkali stress treatment,the Ca²⁺content in P.tenuiflora roots significantly increased,indicating that Ca²⁺may play an important role in P.tenuiflora roots’resistance to alkali stress.Furthermore,using confocal laser scanning microscopy-paraffin-embedded tissue section method,it was observed that alkali stress treatment increased Ca²⁺signal intensity in the cortical cells,endodermal cells,pericycle cells,and xylem cells of P.tenuiflora,indicating that P.tenuiflora may activate multiple Ca²⁺signaling systems in various types of root cells to cope with alkali stress.Transcriptomic analysis showed that one calcium-binding protein gene（CML）,two calcium-dependent protein kinase B genes（CBL）,and four calcium-dependent protein kinase B interacting protein kinase genes（CIPK）were up-regulated in P.tenuiflora roots after alkali stress treatment,indicating that alkali stress can promote the activation of calcium signaling network in P.tenuiflora roots.To verify whether Ca²⁺is involved in Na⁺detoxification in P.tenuiflora roots under alkali stress,15 m M Ca Cl₂was externally applied to P.tenuiflora roots treated with alkali stress for 24 hours.The results showed that external Ca Cl₂treatment significantly reduced the Na⁺level in P.tenuiflora root cells under alkali stress,fully demonstrating that Ca²⁺can participate in Na⁺detoxification in P.tenuiflora roots under alkali stress.（3）After alkali stress treatment,1 CAT,7 GST,and 8 POD were upregulated in the roots of P.tenuiflora,indicating that P.tenuiflora increases the expression levels of antioxidant enzyme genes and promotes the accumulation of antioxidant enzymes in the roots.In addition,some non-enzyme substances also participate in the scavenging of reactive oxygen species under alkali stress conditions.By using confocal laser scanning microscopy in combination with fluorescent dyes,it was found that alkali stress promotes the accumulation of flavonols in the roots of P.tenuiflora.Transcriptome analysis showed that the key genes involved in flavonol synthesis,such as PAL,C4H,4CL,and FLS,were upregulated after alkali stress treatment.These results suggest that under alkali stress,P.tenuiflora may increase its survival rate by regulating gene expression to simultaneously improve the level of antioxidant enzymes and non-enzyme antioxidants,thereby efficiently scavenging ROS.