Physiological Mechanism Of Proline Improving Maize Tolerance To Low Temperature Stress

Maize（Zea mays L.）is the world’s largest food crop and China’s most important food,feed and industrial raw material.Asa thermophilic crop,maize is sensitive to low temperature stress.Heilongjiang Province is the main maize production area in China.T Low temperature stress often occurs in early spring,which seriously affects seed germination,reduces the emergence rate,and ultimately reduces yields.Therefore,improving the cold tolerance during the germination of maize seeds is of great significance for ensuring the yield of maize.Proline is not only an important osmotic adjustment substance,but also plays an important role in regulating plant resistance.However,the response mechanism of proline to low temperature stress during maize seed germination is still unclear.In this study,the maize varieties Xinxin 2（low-temperature insensitive type）and Damin 3307（low-temperature sensitive type）were used as experimental materials for germination test.The germination at room temperature（22℃/10℃,9 d）and low temperature（4℃/4℃,5 d and then transferred to 22℃/10℃,4 d）were set,and the seeds were soaked with the optimal proline concentration(15 mmol·L^-1)obtained from the screening test to study the effects of exogenous proline on the osmotic adjustment system,antioxidant protection system,respiratory metabolism,proline metabolism and endogenous plant hormone content of maize embryos under low temperature stress.At the same time,combined with metabolomics technology,the differential metabolites and differential metabolic pathways regulated by proline were obtained,in order to preliminarily reveal the regulatory basis of exogenous proline on maize embryo response to low temperature stress.Then,the effects of proline on seedling emergence rate,plant height,photosynthesis,nitrogen metabolism,dry matter accumulation,ear traits and yield of maize under low temperature stress associated with early sowing were studied with Damin 3307 as experimental material.To explore the physiological mechanism of exogenous proline improving maize tolerance to low temperature stress.The main results are as follows:（1）Exogenous proline soaking treatment could significantly improve the germination potential,germination rate,germination index and vigor index of two varieties of maize seeds under low temperature stress,increase the length of coleoptile and radicle,increase the dry and fresh weight of young buds（coleoptile and radicle）,and increase the activity ofα-amylase.Among them,15mmol·L^-1proline has the best soaking effect.The results showed that the suitable concentration of exogenous proline could effectively alleviate the inhibition of low temperature stress on maize se ed germination and bud growth,and the alleviation effect on Damin 3307 was more obvious.（2）The activities of superoxide dismutase（SOD）,peroxidase（POD）,ascorbic acid peroxidase（APX）and glutathione reductase（GR）were induced to some extent under lo w temperature stress.The activities of SOD,POD,APX and GR were further enhanced in the soaking seeds with exogenous proline.Exogenous proline treatment improved the activities of catalase（CAT）,monodehydrated ascorbic acid reductase（MDHAR）and dehydroascorbic acid（DHAR）,increased the contents of reduced ascorbic acid（ASA）and reduced glutathione（GSH）,and decreased the contents of oxidized ascorbic acid（DHA）and oxidized glutathione（GSSG）under low temperature stress.The ratio of ASA/DHA and GSH/GSSG increased.The increase in antioxidant enzyme activity and the content of antioxidant can help to maintain the stability of ASA-GSH cycle,and effectively reduce the production rate of superoxide anion（O₂·^-）,hydrogen peroxide（H₂O₂）and malondialdehyde（MDA）In addition,it can also improve the cold tolerance of plants and accelerate the germination of seeds after relieving the stress.（3）Exogenous proline enhanced the activities ofΔ¹-pyrroline-5-carboxylic acid synthetase（P5CS）and ornithine aminotransferase（OAT）in maize embryos under low temperature stress,enhanced the two proline synthesis pathways（glutamate synthesis pathway and ornithine synthesis pathway）,and further enhanced proline accumulation.Exogenous proline treatment induced the activity of proline dehydrogenase（ProDH）in the early stage of low temperature stress and stress relief,which accelerated the degradation of proline,provided a large amount of carbon and nitrogen sources and reducing power for the early stage of embryo adaptation to low temperature stress and rapid seed germination after stress relief,and this effect on Damin 3307 was more obvious.On the other hand,proline,soluble sugar and soluble protein were accumulated in large quanti ties under low temperature stress.Exogenous proline treatment could further promote the accumulation of these osmotic adjustment substances and improve the adaptability of maize embryo to low temperature stress.（4）Under low temperature stress,the activities of hexokinase（HXK）,phosphofructokinase（PFK）,pyruvate kinase（PK）,isocitrate dehydrogenase（IDH）,succinate dehydrogenase（SDH）,malate dehydrogenase（MDH）,glucose-6-phosphate dehydrogenase（G-6-PDH）and 6-phosphogluconate dehydrogenase（6-P-GDH）in maize embryos were decreased,resulting in the decrease of the glycolysis（EMP）pathway,tricarboxylic acid（TCA）cycle and pentose phosphate pathway（PPP）.Soaking seeds with exogenous proline alleviated the inhibition of key enzyme activities of EMP pathway,TCA cycle and PPP of maize embryos under low temperature stress,increased the activities of cytochrome oxidase（COX）and alternative oxidase（AOX）,increased ATP content,alleviated the inhibition of low temperature stress on main cytochrome res piratory pathway(ρV_cyt)activity,and further increased the total respiratory（V _t）and the actual operational activity of alternative pathway(ρV_alt)during seed germination at the initial stage of low temperature stress,improved the inhibition of V_tandρV_alt in the middle and late stage of low temperature stress,and the promotion in Damin 3307 was more significantly.These results indicated that exogenous proline treatment could improve the activity of AOX,alleviate the decrease of COX activity,reduc e electron leakage,reduce the accumulation of ROS,protect the integrity of mitochondrial membrane,partially restore the operation of EMP-TCA pathway,improve PPP efficiency,increase the production of ATP,enhance the respiration during seed germination under low temperature stress and stress relief,and provide raw materials and energy for mitochondrial repair and biosynthesis,and then promote seed germination.（5）Under low temperature stress,the content of abscisic acid（ABA）increased significantly,while gibberellin（GA）,auxin（IAA）and zeatin nucleoside（ZR）decreased significantly,and the disorder degree of endogenous hormones in Damin 3307 was higher than that in Xinxin 2.Seed soaking with exogenous proline could alleviate the decrease of IAA,ZR and GA contents in germinated maize embryos under low temperature stress,reduce the increase of ABA content,improve the cold resistance of maize embryos,alleviate the inhibition of low temperature on seed germination,and promote seed germination after stress relief.（6）Based on ultra-performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）detection platform and self-built database,a total of 589 metabolites were detected in each treated maize embryo.After data processing,262 differential metabolites were obtained,including32 organic acids,28 amino acids,20 nucleotides and derivatives,26 sugars and alcohols,46 lipids,51 alkaloids,44 phenols and 15 other metabolites.68 metabolic pathways involving different metabolites were obtained by KEGG enrichment analysis.The results showed that exogenous proline increased the accumulation of sucrose,raffinose,erythrose-4-phosphate and other sugars and alcohols in response to low temperature stress,increased the content of osmore gulation substances under low temperature stress,and improved the efficiency of EMP and PPP.Exogenous proline also restored TCA cycle by increasing the content ofα-ketoglutarate,succinic acid and fumaric acid.Exogenous proline increased the contents of some amino acids（ornithine,proline,glycine,etc.）,alkaloids（cocamidopropyl betaine,vanillylamine,6-hydroxynicotinic acid,etc.）,phenols（ayapin,chlorogenic acid,etc.）and vitamins（ascorbic acid,etc.）in the embryo under low temperature stress,which were closely related to enhancing the cold resistance of embryo.Combined with pathway enrichment analysis,exogenous proline could enhance the cold resistance of germinated maize embryos by enhancing starch and sucrose metabolism,arginine and pro line metabolism,biosynthesis of secondary metabolites,flavonoid biosynthesis and pentose phosphate pathway.（7）Under the field conditions,exogenous proline(15 mmol·L^-1)soaking treatment could improve the emergence rate of early sowing maize and alleviate the population reduction caused by low temperature.The treatment of seed soaking with proline could significantly increase the plant height at seedling stage and jointing stage,and promote the plant growth.Seed soaking with exogenous proline could effectively regulate the activities of nitrate reductase（NR）,glutamine synthetase（GS）,glutamate synthase（GOGAT）,glutamate dehydrogenase（GDH）,glutamic oxaloacetic transaminase（GOT）and glutamic pyruvic transaminase（GPT）,so as to promote ammonia assimilation,accelerate the synthesis and transformation of protein and amino acid,accelerate plant growth and development,and alleviate the inhibition of nitrogen metabolism caused by early sowing.Meanwhile,early sowing inhibited the activities of net photosynthetic rate（P_n）,stomatal conductance（G_s）,transpiration rate（T_r）,intercellular CO₂ concentration（C_i）,ribose 1,5-diphosphate carboxylase/oxygenase（Ru BPCase）and phosphoenolpyruvate carboxylase（PEPCase）in maize leaves.Seed soaking with exogenous proline could alleviate the decrease of P_n,T_r,G_s and C_i in maize leaves and improve the activity of photosynthetic enzymes.The results showed that the early sowing plants treated with proline could maintain high chlorophyll content（SPAD value）,increase photosynthetic green leaf area,delay leaf senescence,provide stable energy and carbon skeleton supply for nitrogen metabolism,alleviate the inhibition of early sowing on maize growth,improve chilling resistance of maize plants,and promote dry matter accumulation.Exogenous proline significantly increased the number of effective panicles and grains pe r panicle,and ultimately increased the yield of early sowing maize.