Physiological And Biochemical Mechanisms For Water,Nitrogen And Elevated CO₂ Impact On Barley And Oats

Soil drought is often accompanied by low soil nitrogen（N）use efficiency,and reasonable water and N management is important to improve crop water and N use efficiency.Stomata regulate the exchange of gases between plants and atmosphere,and they are important in controlling the two physiological processes of photosynthesis and transpiration.Therefore,exploring the regulatory mechanism of stomatal movement is of great significance to understand the response of crops to environmental stress.In recent years,the atmospheric CO₂ concentration has been increasing.Under the condition of climate change in the future,how to manage water and fertilizer reasonably will be a new problem and challenge in agricultural production.Therefore,in the present study,oat and barley（WT and its correspondent ABA-deficient mutant barley Az34）were used as plant materials,and 15 N,13C,18 O isotope techniques were used to analyze the phenotypic and physiological/biochemical changes of oat under drought and N deficiency stress;the ion fluxes of barley mesophyll/ guard cells were measured by noninvasive micro-test technology（NMT）to quantitatively record and the transmembrane movement of ions,and the mechanisms of phenotypic changes were explored in subcellular and molecular levels combining with the proteomic analysis of leaves;the physiological response of barley to N-fertigation and its behind physiological mechanisms were studied at elevated CO₂,and the quality attributes of barley seeds were analyzed,which would provide theoretical basis for achieving water-saving,fertilizer-saving,high-quality and high-yield agriculture under elevated atmospheric CO₂ concentration and water shortage in the future.The main results are outlined as follows:（1）Moderate drought stress（50% of soil water holding capacity）or high N supply（298 mg/kg N）significantly reduced stomatal conductance,water consumption and increased water use efficiency（WUE）at stomatal and plant levels.Under drought stress or high N treatment,the partial stomatal closure had no significant effect on photosynthetic rate.Although the shoot dry biomass decreased by6.7% or even significantly reduced by 21.3% in the mildly（70% of soil water holding capacity）and moderately drought-stressed treatments,plant water use efficiency（WUEb）of oat plants in these two water deficit treatments increased by 10.8% and 7.4%,respectively,as compared with the well-watered（90% of soil water holding capacity）plants.Moderate drought stress or high N treatment increased shoot carbon isotope composition（δ13C）.At the same time,the positive correlation between δ13C and oxygen isotope composition（δ18O）indicated that the improvement of WUE at stomatal and plant levels under the reduced soil water contents or high N treatment were mainly attributed to the reduction of stomatal conductance.（2）Leaf ABA concentration（[ABA]leaf）of both genotypes increased significantly and WT had a higher [ABA]leaf than Az34 at 2 hours after exposure to 10% polyethylene glycol（PEG）6000.[ABA]leaf of WT was significantly greater compared to that of the control plants while this was not the case for Az34 on 9 d after PEG treatment.The K+ efflux of guard cells was significantly greater in WT than in Az34 at 24 h after PEG treatment.Compared to the control,a significant increase in Ca2+ influx in both genotypes was observed after 2 h exposure to PEG,and which reached the largest value after 4 h in WT.The increase of [ABA]leaf coincided with the increase of K+ efflux and Ca2+ influx and the decrease of stomatal conductance in WT under short-term drought stress,though the concentrations of IAA,GA3 and ZR in WT were all increased at 4 h.In addition,a large H+ influx in leaf mesophyll could cause apoplastic alkalization facilitating the transport of xylem-borne ABA to guard cells.These results elucidate the mechanism of ABA regulating stomatal movement by mediating ion transport in guard cells under PEG-induced drought stress.（3）When plants grew under without PEG（P0）,[ABA]leaf of barley plants exposure to Hoagland’s nutrient solution without N（N0）for 72 h were higher than those of plants grown under Hoagland’s nutrient solution（N1）,while stomatal conductance and transpiration rate of N0 plants were slightly lower than that of N1 plants,but there was no significant difference.Under P0,the photosynthetic rate of two genotypes barley decreased significantly under N0 for 72 h while there was no significant difference between N0 and N1 plants under 15% PEG treatment（P15）.Regardless of N treatment,the photosynthetic rate of P15 plants was lower than that of P0 plants.The photosynthetic rate under N0 treatment for 72 h was significantly lower than that under N0 treatment for 12 h.Drought stress（12 h or72 h）significantly reduced the root water potential of barley.Under P0 for 72 h,the root water potential of N0 plants was higher than that of N1 plants.The results showed that the K+ efflux was caused by PEG induced drought stress（12 h or 72 h）,which was consistent with the decrease of stomatal conductance and ABA.At the same time,the K transporter protein of WT leaves was down regulated under N deficiency,drought stress or the interaction between drought stress and N deficiency.WT could enhance the adaptability to environmental stress（N deficiency,drought stress or the interaction between drought stress and N deficiency）by strengthening glycolysis pathway and osmotic regulation.（4）The two genotypes showed different responses to reduced fertigation regimes,especially at elevated CO₂ concentration（e[CO₂],800 ppm）.Although e[CO₂] had little effect on stomatal conductance,transpiration rate as well as plant water use of WT,especially under N-fertigation at reduced irrigation volume treatment（DIN,where 70% of water consumption of FIN was irrigated to the whole pot）and PRDN（alternate N-fertigation at reduced irrigation volume,where 70% of water consumption of FIN was irrigated to only half of the pot until the soil water content of the dry side decreased to 7%-10%,then the irrigation was shifted to the previously dried side）,it increased photosynthetic rate,resulting in an increased WUE at stomatal,leaf and whole plant levels.For Az34,the positive effect of e[CO₂] on WUE was attributed to both significantly enhanced photosynthetic rate and lowered stomatal conductance and transpiration rate.For both genotypes,e[CO₂] increased100-grain weight and shoot dry biomass but didn’t affect grain yield and WUE for grain production（WUEg）.PRDN increased grain yield,harvest index and WUEg of both genotypes regardless of [CO₂],compared to FIN（N-fertigation at full irrigation volume,in which the soil water holding capacity was maintained at 90%）.DIN and PRDN increased N uptake of both genotypes at e[CO₂],compared to FIN.Compare to ambient CO₂ concentration（a[CO₂],400 ppm）,e[CO₂] increased 15 N uptake and 15 N recovery rate of both genotypes by enhancing biomass accumulation.In addition,both genotypes grown under DIN and PRDN allocated more N to the grain,compared to the FIN plants.Collectively,N-fertigation at reduced irrigation volume promoted N allocation to the grain and increased WUE,particularly under e[CO₂].Reduced irrigation regimes,especially PRDN,are recommended for optimizing WUE and N nutrition of crops in a future water-limited and CO₂-enriched environment.（5）e[CO₂] alleviated the negative effects of water deficit on N,P and S concentrations,C/N ratio and C/P ratio of barley grain.The concentrations of K,Ca,Mg,Fe,Mn and Cu in barley grain were not affected by DIN and PRDN treatments.However,the accumulation of K,Ca,Mg,Fe,Mn and Cu increased correspondingly due to the increase of barley grain yield under DIN and PRDN treatments.e[CO₂] increased Fe and Cu concentrations of grain,and B concentration of WT grain was increased under DIN and PRDN treatments.The concentrations of P,K,Ca,Mg,S,Mn and Zn were not affected by [CO₂].Under e[CO₂] in the future,DIN and PRDN had the potential to become a water-saving and high-quality irrigation method.