Physiological And Biochemical Mechanisms Of Maize Seedlings Response To Cold Stress Under Different Nitrogen Supply Conditions

The global population of human beings is expected to reach 9.8 billion by 2050,highlighting the urgent need to provide adequate food and nutrition to keep pace with population growth.However,abiotic stressors such as cold stress(CS)critically impacts plant growth and development,and reduces crop productivity worldwide.Maize,an essential staple food and feed,known as the third largest cultivated grain crops,is particularly susceptible to CS due to its inherent sensitivity as a tropical/subtropical crop.CS adversely affects many physiological and biochemical processes,which determine the sensitivity level of maize plant to the stress.In northwestern and eastern regions of China and central Europe,maize cultivation frequently encounters CS during the spring sowing stage and early growth stage,which mainly inhibits maize seedling establishment and growth at the seedling stage,subsequently limiting the agricultural production and inducing a critical economic loss.Elsewhere,nitrogen(N)is an essential macronutrient largely required for maize plant growth and development owing to its participation in the biosynthesis of essential macromolecules(proteins,hormones,amino acids,and energy transfer molecules).N metabolism is crucial for most stress-responsive physiological and biochemical processes in maize.The alleviation of stress effects through applying appropriate N levels is already known in various crops and stress types;however,the particular regulating mechanisms are still unknown and scarcely reported in maize plants exposed to CS.Hence,to decipher how N interacts with CS while modulating maize growth and development,we performed physiological,biochemical,and hormonal analyses associated with N metabolism in response to 3 different N levels[high(HN)at 8 m M,medium(MN)at 4 m M,and low(LN)at 2 m M of NH4~+and NO3~-]applied during prestress stage(4 d),under CS(8/4?C,4 d)and following recovery from cold(28/22?C,4 d)of maize plant grown hydroponically as designed for experiment 1,while during experiment 2,N was only applied during recovery after CS.From the results obtained in both experiments(1 and 2),we observed that CS did adversely affect maize seedlings growth and development,as reflected by significant reduction(P<0.05)in plant height(15%),leaf area,root length and area,fresh weight and dry matter of shoot and root(50%average reduction)relative to control.Similarly,CS induced leaf wilting and chlorosis which resulted in 20%total chlorophyll pigments reduction relative to control,indicating a higher sensitivity of maize seedlings to temperature stress.In both experiments(1 and 2),chlorophyll fluorescence analysis indicated that CS significantly inhibited the activity of photosystem I(PSI)and photosystem II(PSII),more significantly PSI due to the limitation of electron transport/availability in both donor and acceptor sides of PSI.With the N application under both experiments,CS-induced photoinhibition was relieved and the activity of PSI and PSII increased,more particularly when N dose increased in HN treatment relative to LN,which enhanced the maximum photochemical efficiency and quantum efficiency of photosystem II,and decreased the thermal dissipation in the PSII antennae to enable the transfer of excitation energy from PSII antennae to PSII reaction centers,thus an enhanced electron transfer rate of PSII.Through the analysis of photosynthetic assimilation and observation of photosynthetic apparatus(stomata,mesophyll and bundle sheath)ultrastructure,we found almost 55%(P<0.05)cold-induced reduction of photosynthetic capacity,which was not only linked to the poor stomatal conductance but also the decrease abundance of photosynthetic enzyme(Rubis CO).An alleviation of these adverse effects was exhibited following N application;Compared to LN,increasing N dose in HN supply stimulated 20%increase of photosynthetic assimilation through an increase of stomatal area and aperture to increase stomatal conductance,and a higher Rubis CO content that promote CO2 assimilation under CS,under both experimental conditions,more so in the first experiment.Based on hormonal analysis in experiment 1,we found a higher accumulation of ABA in cold-treated maize leaves,indicating the induction of ABA function in maize response to CS,thus revealing an ABA-dependent pathway in maize seedlings.Similarly,our results showed that increasing N supply in HN decreased ABA accumulation compared to LN,which was linked to the increase of stomatal opening,implying that HN supply reduced stomatal sensitivity to ABA under CS.In this study,under experiment 1 and 2,our results revealed and ascertained that CS is a limiting factor of N metabolism(N uptake,transport,and content)in maize at seedling stage.Further,we demonstrated that the impairment of N metabolism in maize was mitigated by the supply of N,and the mitigating effect increases as N concentration increased in HN treatment relative to LN,as showed by higher N uptake and transport(average increase of 40-50%),contents of total N and nitrate(average increase of 25-30%),and activity of N-assimilating enzymes:nitrate reductase(40%increase),nitrite reductase(93%increase),glutamine synthase(69%increase),and glutamate synthase(23%increase),particularly under experiment1.Importantly,these results displayed that the induced growth,biomass,photosynthesis,and N metabolism in HN-treated plants can be used as evaluating factors of CS tolerance in and recovery ability of maize.In this study,we observed that growth and photosynthetic parameters,ROS homeostasis,and N assimilation were relatively increased when N dose increases in HN either under continuous supply of N from cold to recovery period(experiment 1)or a sole supply of N during recovery from cold(experiment2).This revealed that N supply not only plays an essential role in mitigating growth inhibitory effects under CS but also boost the recovery ability of maize plants after CS.